Methods of modulating tubulin deacetylast activity

ABSTRACT

The present invention provides methods for identifying agents that modulate a level or an activity of tubulin deacetylase polypeptide, as well as agents identified by the methods. The invention further provides methods of modulating tubulin deacetylase activity in a cell. The invention further provides methods of modulating cellular proliferation by modulating the activity of tubulin deacetylase.

CROSS REFERENCE

This application is a divisional of U.S. patent application Ser. No.10/441,854, filed May 19, 2003, which application claims the benefit ofU.S. Provisional Patent Application No. 60/382,218, filed May 20, 2002,which applications are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention is in the field of deacetylase enzymes, andenzymes that modify tubulin.

BACKGROUND OF THE INVENTION

Reversible histone acetylation is under the control of opposingenzymatic activities of two categories of enzymes: histone deacetylases(HDACs) and histone acetyltransferases (HATs). Deacetylation of lysineresidues on N-terminal tails of histones by HDACs is generallyassociated with transcriptional silencing, whereas acetylation of thesame lysine residues is associated with transcriptional activation. Inaddition to histones, a rapidly growing number of other non-histoneproteins undergo the post-translational modification of acetylation onlysine residues. An example of some of these proteins include HMG-14 and17, HMGI(Y), p53, E2F1, NF-κB, and the HIV-1 Tat protein. HDACs areseparated into three distinct classes based on their homology to yeasttranscriptional repressors. Class I and Class II deacetylases arehomologues of the Rpd3p and Hda1p proteins, respectively. Class IIIHDACs are defined based on their homology to the yeast transcriptionalrepressor, Sir2p.

The Silent Information Regulator (SIR) gene family was initiallyidentified based on its role in the regulation of gene expression at theHM loci in S. cerevisiae. Later studies further defined the role of SIRproteins in transcriptional silencing at a number of additional loci inthe yeast genome, including telomeres, rDNA locus, and at sites of DNAdamage. Silencing at the telomeres and the HM loci, is mediated by amulti-protein complex which includes Sir2p, Sir3p and Sir4p, with Sir1pbeing involved in silencing at the HM loci only. Interestingly,silencing and repression of recombination at the rDNA locus is achievedby Sir2p in association with the RENT complex, containing Net1, Nan1 andcdc14, and has been associated with aging in S. cerevisiae. The recentdiscovery that SIR2 encodes an NAD-dependent histone deacetylase hasvalidated the long held suspicion that this protein regulated the levelof histone acetylation.

The SIR2 family of genes is conserved from archaebacteria to eukaryotes.In S. cerevisiae, this family consist of Sir2 and four closely relatedgenes (HST1-4). Whereas Sir2p and HST1p are localized primarily in thenucleus, Hst2p is exclusively cytoplasmic. Humans have seven proteinswith homology to the S. cerevisiea Sir2p, which have been named Sirtuinsor SIRTs. Human SIRT1 and mouse Sir2α, which are most closely homologousto Sir2p and HST1p, exhibit protein deacetylase activity withspecificity for the transcription factor protein p53. Deacetylation ofp53 by SIRT1 suppresses p53-dependent apoptosis in response to DNAdamage. The human SIRT2 protein, which is most closely related to Hst2p,is also localized in the cytoplasm. Interestingly, both SIRT2 and Hst2pregulate rDNA and telomeric silencing indirectly from their cytoplasmiclocation.

The microtubule network is formed by the polymerization of α/β tubulinheterodimers and plays an important role in the regulation of cellshape, intracellular transport, cell motility, and cell division. α andβ tubulin sub-units are subject to numerous post-translationalmodifications including tyrosination, phosphorylation,polyglutamylation, polyglycylation and acetylation. Tubulin representsone of the major acetylated cytoplasmic proteins. Acetylation of tubulintakes place on lysine-40 of α-tubulin, which based on the crystalstructure of the tubulin heterodimer, is predicted to lie within theluminal side of the polymerized microtubule.

A variety of physiological signals have been reported to modulate thelevel of tubulin acetylation. This includes the anticancer drugpaclitaxel, as well as association of MAP1 and 2C, tau, and the herpessimplex virus encoded protein VP22. Similarly, microtubules associatedwith stable structures, such as cilia, contain relativelyhyperacetylated α-tubulin. These observations have supported the notionthat stabilized microtubules become hyperacetylated. However, theenzymes responsible for the reversible acetylation of tubulin have notbeen identified. This lack of reagents has precluded a thorough analysisof the biological role of tubulin acetylation in microtubule dynamics,stability and physiological functions of the cytoskeleton.

Literature

-   Frye (1999) Biochem. Biophys. Res. Comm. 260:273-279; Smith et    al. (2000) Proc. Natl. Acad. Sci. USA 97:6658-6663; Landry et    al. (2000) Biochem. Biophys. Res. Comm. 278:685-690; Tanner et    al. (2000) Proc. Natl. Acad. Sci. USA 97:14178-14182; Finnin et    al. (2001) Nat. Struct. Biol. 8:621-625; MacRae (1997) Eur. J.    Biochem. 244:265-278; North et al. (2003) Mol. Cell. 11:437-444.

SUMMARY OF THE INVENTION

The present invention provides methods for identifying agents thatmodulate a level or an activity of tubulin deacetylase polypeptide, aswell as agents identified by the methods. The invention further providesmethods of modulating tubulin deacetylase activity in a cell. Theinvention further provides methods of modulating cellular proliferationby modulating the activity of tubulin deacetylase.

FEATURES OF THE INVENTION

The invention features an in vitro method of identifying an agent thatmodulates an enzymatic activity of a human tubulin deacetylase, e.g.,human SIRT2. The method generally comprises contacting a tubulindeacetylase polypeptide with a test agent in an assay mixture thatcomprises nicotinamide adenine dinucleotide (NAD) and an acetylatedtubulin peptide; and determining the effect, if any, of the test agenton the enzymatic activity of tubulin deacetylase. In some embodiments,the tubulin deacetylase polypeptide comprises an amino acid sequence asset forth in SEQ ID NO:02. In some embodiments, the acetylated tubulinpeptide comprises the sequence NH₂-MPSD(AcK)TIGG-CO₂ (SEQ ID NO:08). Insome embodiments, the acetylated tubulin peptide contains a ¹⁴C-labeledacetyl group on a lysine corresponding to Lys-40 of native tubulin, anddetermination of the effect of the agent on the enzyme is by measuringrelease of the radioactive acetyl group. In some embodiments, the effectof the agent on the activity of the enzyme is by detecting binding of anantibody specific for acetylated tubulin.

The present invention further features an in vitro method foridentifying an agent that modulates a level of tubulin deacetylase in acell. The method generally involves contacting a cell that producestubulin deacetylase with a test agent; and determining the effect, ifany, of the test agent on the level of tubulin deacetylase. In someembodiments, determining the effect of the agent involves determining alevel of tubulin deacetylase mRNA in the cell. In other embodiments,determining the effect of the agent involves determining a level oftubulin deacetylase polypeptide in the cell.

The present invention further features a biologically active agentidentified by a method according to the invention. The present inventionfurther features a pharmaceutical composition comprising a biologicallyactive agent that reduces a level or an activity of a tubulindeacetylase protein; and a pharmaceutically acceptable excipient. Thepresent invention further features a method of modulating cellproliferation, the method comprising contacting a cell with an agent ofthe invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D depict NAD-dependent deacetylation of a histone peptide byhuman SIRT2.

FIGS. 2A-C depict inactivation of SIRT2 histone deacetylase activity bypoint mutations within the SIRT2 catalytic domain.

FIGS. 3A-E depict SIRT2 tubulin deacetylates tubulin ex vivo.

FIGS. 4A-D depict the substrate preference for SIRT2.

FIGS. 5A and 5B depict regulation of MIZ-1 sub-cellular distribution byacetylated tubulin.

FIGS. 6A and 6B depict the nucleotide and amino acid sequences,respectively, of human SIRT2 (SEQ ID NOs:01 and 02, respectively).

FIG. 7 depicts ribavirin inhibition of SIRT2.

DEFINITIONS

The terms “polypeptide” and “protein”, used interchangeably herein,refer to a polymeric form of amino acids of any length, which caninclude coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;and the like.

A “substantially isolated” or “isolated” polypeptide is one that issubstantially free of the macromolecules with which it is associated innature. By substantially free is meant at least 50%, preferably at least70%, more preferably at least 80%, and even more preferably at least 90%free of the materials with which it is associated in nature.

A “biological sample” encompasses a variety of sample types obtainedfrom an individual and can be used in a diagnostic or monitoring assay.The definition encompasses blood and other liquid samples of biologicalorigin, solid tissue samples such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples.

The terms “cancer”, “neoplasm”, “tumor”, and “carcinoma”, are usedinterchangeably herein to refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation.Cancerous cells can be benign or malignant.

As used herein, the terms “treatment”, “treating”, and the like, referto obtaining a desired pharmacologic and/or physiologic effect. Theeffect may be prophylactic in terms of completely or partiallypreventing a disease or symptom thereof and/or may be therapeutic interms of a partial or complete cure for a disease and/or adverse affectattributable to the disease. “Treatment”, as used herein, covers anytreatment of a disease in a mammal, particularly in a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “atubulin deacetylase” includes a plurality of such deacetylases andreference to “the agent” includes reference to one or more agents andequivalents thereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of identifying an agent thatmodulates a level or an activity of tubulin deacetylase; agentsidentified by the methods; and therapeutic methods, including methods ofstabilizing microtubules, methods of controlling unwanted cellularproliferation, and methods of treating disorders associated with orcaused by unwanted cellular proliferation.

The invention is based in part on the observation that human SIRT2 is anNAD-dependent tubulin deacetylase. The human SIRT2 enzyme is acytoplasmic protein that is closely related to the Saccharomycescerevisiae protein Hst2P, which does not deacetylate tubulin; and is anortholog of the S. cerevisiae Silent Information Regulator 2 protein(Sir2p), a histone deacetylase that plays a role in transcriptionalsilencing. Human SIRT2 removes an acetyl group from lysine-40 ofα-tubulin. Deacetylation of tubulin also results in a reduction in thespecific interaction of tubulin with the transcription factormyc-interacting zinc finger-1 (“MIZ-1”).

Identification of human SIRT2 as a tubulin deacetylase alloweddevelopment of assays to identify agents that modulate the activity ofthis enzyme. Agents that modulate the level or the enzymatic activity ofhuman SIRT2 are useful for modulating cellular proliferation, and aretherefore useful, e.g., as anti-cancer agents.

Screening Methods

The invention provides in vitro methods of identifying an agent thatmodulates a level or an activity of a tubulin deacetylase. The methodsgenerally involve contacting a tubulin deacetylase protein, or a cellthat produces a tubulin deacetylase protein, with a test agent, anddetermining the effect, if any, on a level or an activity of the tubulindeacetylase protein.

In some embodiments, the methods are cell-free methods. Cell-freemethods generally involve contacting a tubulin deacetylase with a testagent and determining the effect, if any, of the test agent on theenzymatic activity of the tubulin deacetylase.

In other embodiments, the methods are cell-based methods. Cell-basedmethods generally involve contacting a cell that produces tubulindeacetylase with a test agent and determining the effect, if any, of thetest agent on the level of tubulin deacetylase mRNA or tubulindeacetylase protein in the cell. In some embodiments, cell-based methodsinvolve contacting a cell that produces tubulin deacetylase with a testagent and determining the effect, if any, of the test agent on thebinding of a protein to tubulin.

As used herein, the term “determining” refers to both quantitative andqualitative determinations and as such, the term “determining” is usedinterchangeably herein with “assaying,” “measuring,” and the like.

The term “tubulin deacetylase polypeptide” encompasses human tubulindeacetylase proteins (e.g., human SIRT2 proteins) having the amino acidsequences set forth in any of GenBank Accession Nos. NM_(—)012237;AF083107; and NM_(—)030593, or depicted in FIG. 6B, where thepolypeptide is a cytoplasmic protein and exhibits NAD-dependent tubulindeacetylase activity. The term encompasses variants that haveinsertions, deletions, and/or conservative amino acid substitutions thatdo not affect the ability of the protein to deacetylate α-tubulin havingan acetylated lysine at position 40. In some embodiments, the tubulindeacetylase is recombinant, e.g., produced in a cell transfected with anexpression construct comprising a nucleotide sequence that encodes thetubulin deacetylase.

The term “tubulin deacetylase polypeptide” further encompasses fusionproteins comprising a tubulin deacetylase and a heterologous polypeptide(“fusion partners”), where suitable fusion partners includeimmunological tags such as epitope tags, including, but not limited to,hemagglutinin, FLAG (see, e.g., Archives of Biochem and Biophys.406:209-221, 2002; J. Bio. Chem., 277(23):20750-20755,2002), and thelike; proteins that provide for a detectable signal, including, but notlimited to, fluorescent proteins (e.g., a green fluorescent protein, afluorescent protein from an Anthozoan species, and the like), enzymes(e.g., β-galactosidase, luciferase, horse radish peroxidase, etc.), andthe like; polypeptides that facilitate purification or isolation of thefusion protein, e.g., metal ion binding polypeptides such as 6H is tags(e.g., tubulin deacetylase/6H is), glutathione-5-transferase (GST), andthe like. The term “tubulin deacetylase polypeptide” further includes atubulin deacetylase polypeptide modified to include one or more specificprotease cleavage sites.

Activities attributed to tubulin deacetylase include acetylation ofLys-40 of tubulin; control of MIZ-1/tubulin binding; and the like. Thus,an “activity of tubulin deacetylase” includes direct activity, e.g.,acetylation of tubulin; and indirect activities, e.g., a reduction inMIZ-1 binding to tubulin. A MIZ-1 protein amino acid sequence is foundunder GenBank Accession No. Q13105.

Where the assay is an in vitro cell-free assay, the methods generallyinvolve contacting a tubulin deacetylase polypeptide with a test agent.The tubulin deacetylase polypeptide may be, but need not be, purified.For example, the tubulin deacetylase polypeptide can be in a celllysate, or may be isolated, or partially purified. Thus, the assay canbe conducted in the presence of additional components, as long as theadditional components do not adversely affect the reaction to anunacceptable degree.

Where the assay is an in vitro cell-based assay, any of a variety ofcells can be used. The cells used in the assay are usually eukaryoticcells, including, but not limited to, rodent cells, human cells, andyeast cells. The cells may be primary cell cultures or may beimmortalized cell lines. The cells may be “recombinant,” e.g., the cellmay have transiently or stably introduced therein a construct (e.g., aplasmid, a recombinant viral vector, or any other suitable vector) thatcomprises a nucleotide sequence encoding a tubulin deacetylasepolypeptide, or that comprises a nucleotide sequence that comprises atubulin deacetylase promoter operably linked to a reporter gene.

The terms “candidate agent,” “test agent,” “agent”, “substance” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.) and can also be used. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available from Pan Labs (Bothell, Wash.) or are readily producible.

Candidate agents may be small organic or inorganic compounds having amolecular weight of more than 50 and less than about 2,500 daltons.Candidate agents may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Assays of the invention include controls, where suitable controlsinclude a sample (e.g., a sample comprising tubulin deacetylase protein,or a cell that synthesizes tubulin deacetylase) in the absence of thetest agent. Generally a plurality of assay mixtures is run in parallelwith different agent concentrations to obtain a differential response tothe various concentrations. Typically, one of these concentrationsserves as a negative control, i.e. at zero concentration or below thelevel of detection.

Where the screening assay is a binding assay (e.g., binding to tubulindeacetylase; MIZ-1 binding to tubulin), one or more of the molecules maybe joined to a label, where the label can directly or indirectly providea detectable signal. Various labels include radioisotopes, fluorescers,chemiluminescers, enzymes, specific binding molecules, particles, e.g.magnetic particles, and the like. Specific binding molecules includepairs, such as biotin and streptavidin, digoxin and antidigoxin etc. Forthe specific binding members, the complementary member would normally belabeled with a molecule that provides for detection, in accordance withknown procedures.

A variety of other reagents may be included in the screening assay.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc that are used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Reagentsthat improve the efficiency of the assay, such as protease inhibitors,nuclease inhibitors, anti-microbial agents, etc. may be used. Thecomponents of the assay mixture are added in any order that provides forthe requisite binding or other activity. Incubations are performed atany suitable temperature, typically between 4° C. and 40° C. Incubationperiods are selected for optimum activity, but may also be optimized tofacilitate rapid high-throughput screening. Typically between 0.1 and 1hour will be sufficient.

The screening methods may be designed a number of different ways, wherea variety of assay configurations and protocols may be employed, as areknown in the art. For example, one of the components may be bound to asolid support, and the remaining components contacted with the supportbound component. The above components of the method may be combined atsubstantially the same time or at different times.

Where the assay is a binding assay, following the contact and incubationsteps, the subject methods will generally, though not necessarily,further include a washing step to remove unbound components, where sucha washing step is generally employed when required to remove label thatwould give rise to a background signal during detection, such asradioactive or fluorescently labeled non-specifically bound components.Following the optional washing step, the presence of bound complexeswill then be detected.

A test agent of interest is one that reduces a level of tubulindeacetylase protein or inhibits a tubulin deacetylase activity by atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 80%, at least about 90%, ormore, when compared to a control in the absence of the test agent.

Methods of Detecting Agents that Modulate a Level of Tubulin DeacetylasemRNA and/or Tubulin Deacetylase Polypeptide

The subject screening methods include methods of detecting an agent thatmodulates a level of a tubulin deacetylase mRNA and/or tubulindeacetylase polypeptide in a cell. In some embodiments, the methodsinvolve contacting a cell that produces tubulin deacetylase with a testagent, and determining the effect, if any, of the test agent on thelevel of tubulin deacetylase mRNA in the cell.

A candidate agent is assessed for any cytotoxic activity it may exhibittoward the cell used in the assay, using well-known assays, such astrypan blue dye exclusion, an MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)assay, and the like. Agents that do not exhibit cytotoxic activity areconsidered candidate agents.

A wide variety of cell-based assays may be used for identifying agentswhich reduce a level of tubulin deacetylase mRNA in a eukaryotic cell,using, for example, a cell that normally produces tubulin deacetylasemRNA, a mammalian cell transformed with a construct comprising a tubulindeacetylase-encoding cDNA such that the cDNA is overexpressed, or,alternatively, a construct comprising a tubulin deacetylase promoteroperably linked to a reporter gene.

Accordingly, the present invention provides a method for identifying anagent, particularly a biologically active agent, that reduces a level oftubulin deacetylase expression in a cell, the method comprising:combining a candidate agent to be tested with a cell comprising anucleic acid which encodes a tubulin deacetylase polypeptide, or aconstruct comprising a tubulin deacetylase promoter operably linked to areporter gene; and determining the effect of said agent on tubulindeacetylase expression. A decrease of at least about 10%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 80%, at least about 90%, or more, in the level (i.e., anamount) of tubulin deacetylase mRNA and/or polypeptide followingcontacting the cell with a candidate agent being tested, compared to acontrol to which no agent is added, is an indication that the agentmodulates tubulin deacetylase expression.

Tubulin deacetylase mRNA and/or polypeptide whose levels are beingmeasured can be encoded by an endogenous tubulin deacetylasepolynucleotide, or the tubulin deacetylase polynucleotide can be onethat is comprised within a recombinant vector and introduced into thecell, i.e., the tubulin deacetylase mRNA and/or polypeptide can beencoded by an exogenous tubulin deacetylase polynucleotide. For example,a recombinant vector may comprise an isolated tubulin deacetylasetranscriptional regulatory sequence, such as a promoter sequence,operably linked to a reporter gene (e.g., β-galactosidase,chloramphenicol acetyl transferase, a fluorescent protein, luciferase,or other gene that can be easily assayed for expression).

In these embodiments, the method for identifying an agent that modulatesa level of tubulin deacetylase expression in a cell, comprises:combining a candidate agent to be tested with a cell comprising anucleic acid which comprises a tubulin deacetylase gene transcriptionalregulatory element operably linked to a reporter gene; and determiningthe effect of said agent on reporter gene expression. A recombinantvector may comprise an isolated tubulin deacetylase transcriptionalregulatory sequence, such as a promoter sequence, operably linked tosequences coding for a tubulin deacetylase polypeptide; or thetranscriptional control sequences can be operably linked to codingsequences for a tubulin deacetylase fusion protein comprising tubulindeacetylase polypeptide fused to a polypeptide which facilitatesdetection. In these embodiments, the method comprises combining acandidate agent to be tested with a cell comprising a nucleic acid whichcomprises a tubulin deacetylase gene transcriptional regulatory elementoperably linked to a tubulin deacetylase polypeptide-coding sequence;and determining the effect of said agent on tubulin deacetylaseexpression, which determination can be carried out by measuring anamount of tubulin deacetylase mRNA, tubulin deacetylase polypeptide, ortubulin deacetylase fusion polypeptide produced by the cell.

Cell-based assays generally comprise the steps of contacting the cellwith an agent to be tested, forming a test sample, and, after a suitabletime, assessing the effect of the agent on tubulin deacetylaseexpression. A control sample comprises the same cell without thecandidate agent added. Tubulin deacetylase expression levels aremeasured in both the test sample and the control sample. A comparison ismade between tubulin deacetylase expression level in the test sample andthe control sample. tubulin deacetylase expression can be assessed usingconventional assays. For example, when a mammalian cell line istransformed with a construct that results in expression of tubulindeacetylase, tubulin deacetylase mRNA levels can be detected andmeasured, or tubulin deacetylase polypeptide levels can be detected andmeasured. A suitable period of time for contacting the agent with thecell can be determined empirically, and is generally a time sufficientto allow entry of the agent into the cell and to allow the agent to havea measurable effect on tubulin deacetylase mRNA and/or polypeptidelevels. Generally, a suitable time is between 10 minutes and 24 hours,or from about 1 hour to about 8 hours.

Methods of measuring tubulin deacetylase mRNA levels are known in theart, several of which have been described above, and any of thesemethods can be used in the methods of the present invention to identifyan agent which modulates tubulin deacetylase mRNA level in a cell,including, but not limited to, a polymerase chain reaction (PCR), suchas a PCR employing detectably labeled oligonucleotide primers, and anyof a variety of hybridization assays.

Similarly, tubulin deacetylase polypeptide levels can be measured usingany standard method, several of which have been described herein,including, but not limited to, an immunoassay such as enzyme-linkedimmunosorbent assay (ELISA), for example an ELISA employing a detectablylabeled antibody specific for a tubulin deacetylase polypeptide.

Tubulin deacetylase polypeptide levels can also be measured in cellsharboring a recombinant construct comprising a nucleotide sequence thatencodes a tubulin deacetylase fusion protein, where the fusion partnerprovides for a detectable signal or can otherwise be detected. Forexample, where the fusion partner provides an immunologicallyrecognizable epitope (an “epitope tag”), an antibody specific for anepitope of the fusion partner can be used to detect and quantitate thelevel of tubulin deacetylase. In some embodiments, the fusion partnerprovides for a detectable signal, and in these embodiments, thedetection method is chosen based on the type of signal generated by thefusion partner. For example, where the fusion partner is a fluorescentprotein, fluorescence is measured.

Fluorescent proteins suitable for use include, but are not limited to, agreen fluorescent protein (GFP), including, but not limited to, a“humanized” version of a GFP, e.g., wherein codons of thenaturally-occurring nucleotide sequence are changed to more closelymatch human codon bias; a GFP derived from Aequoria Victoria or aderivative thereof, e.g., a “humanized” derivative such as Enhanced GFP,which are available commercially, e.g., from Clontech, Inc.; a GFP fromanother species such as Renilla reniformis, Renilla mulleri, orPtilosarcus guernyi, as described in, e.g., WO 99/49019 and Peelle etal. (2001) J. Protein Chem. 20:507-519; “humanized” recombinant GFP(hrGFP) (Stratagene); any of a variety of fluorescent and coloredproteins from Anthozoan species, as described in, e.g., Matz et al.(1999) Nature Biotechnol. 17:969-973; and the like. Where the fusionpartner is an enzyme that yields a detectable product, the product canbe detected using an appropriate means, e.g., β-galactosidasecan,depending on the substrate, yield colored product, which is detectedspectrophotometrically, or a fluorescent product; luciferase can yield aluminescent product detectable with a luminometer; etc.

A number of methods are available for analyzing nucleic acids for thepresence and/or level of a specific mRNA in a cell. The mRNA may beassayed directly or reverse transcribed into cDNA for analysis. Thenucleic acid may be amplified by conventional techniques, such as thepolymerase chain reaction (PCR), to provide sufficient amounts foranalysis. The use of the polymerase chain reaction is described inSaiki, et al. (1985), Science 239:487, and a review of techniques may befound in Sambrook, et al. Molecular Cloning: A Laboratory Manual, CSHPress 1989, pp. 14.2-14.33. Alternatively, various methods are known inthe art that utilize oligonucleotide ligation as a means of detectingpolymorphisms, for examples see Riley et al. (1990), Nucl. Acids Res.18:2887-2890; and Delahunty et al. (1996), Am. J. Hum. Genet.58:1239-1246.

A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2′,7′-dimethoxy-4′,5′-dichloro-6-carboxyfluorescein (JOE),6-carboxy-X-rhodamine (ROX),6-carboxy-2′,4′,7′,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, 3S, 3H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

A variety of different methods for determining the nucleic acidabundance in a sample are known to those of skill in the art, whereparticular methods of interest include those described in: Pietu et al.,Genome Res. (June 1996) δ: 492-503; Zhao et al., Gene (Apr. 24, 1995)156: 207-213; Soares, Curr. Opin. Biotechnol. (October 1997) 8: 542-546;Raval, J. Pharmacol Toxicol Methods (November 1994) 32: 125-127;Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216: 299-304; Stolz &Tuan, Mol. Biotechnol. (December 19960 6: 225-230; Hong et al.,Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143:298. Also of interest are the methods disclosed in WO 97/27317, thedisclosure of which is herein incorporated by reference.

A number of methods are available for determining the expression levelof a gene or protein in a particular sample. For example, detection mayutilize staining of cells or histological sections with labeledantibodies, performed in accordance with conventional methods. Cells arepermeabilized to stain cytoplasmic molecules. The antibodies of interestare added to the cell sample, and incubated for a period of timesufficient to allow binding to the epitope, usually at least about 10minutes. The antibody may be labeled with radioisotopes, enzymes,fluorescers, chemiluminescers, or other labels for direct detection.Alternatively, a second stage antibody or reagent is used to amplify thesignal. Such reagents are well known in the art. For example, theprimary antibody may be conjugated to biotin, with horseradishperoxidase-conjugated avidin added as a second stage reagent. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. Alternatively, the secondary antibody conjugated to afluorescent compound, e.g. fluorescein, rhodamine, Texas red, etc. Theabsence or presence of antibody binding may be determined by variousmethods, including flow cytometry of dissociated cells, microscopy,radiography, scintillation counting, etc.

Methods of Detecting Agents that Modulate an Activity of a TubulinDeacetylase Polypeptide

Methods of detecting an agent that modulates an activity of a tubulindeacetylase polypeptide include cell-free and cell-based methods. Themethods generally involve contacting a tubulin deacetylase polypeptidewith a test agent and determining the effect, if any, on the tubulindeacetylase enzyme activity.

Methods of assaying tubulin deacetylase enzyme activity are known in theart, and any known method can be used. As one non-limiting example, anacetylated tubulin peptide is incubated, together with NAD, with thetubulin deacetylase and a test agent, and the effect, if any, of thetest agent on deacetylation of the tubulin peptide is determined.Acetylated tubulin peptides generally comprise an amino acid sequencethat comprises the Lys-40 of native tubulin, plus three, four, five,six, seven, or more, amino acids on the NH₂ terminal side, and three,four, five, six, seven, or more, amino acids on the CO₂ terminal side ofthe Lys-40 of native tubulin. As one non-limiting example, an acetylatedtubulin peptide has the sequence NH₂-MPSD(AcK)TIGG-CO₂ (SEQ ID NO:08).Those skilled in the art can readily design additional acetylatedtubulin peptides. The acetylated tubulin peptide is present in the assaymixture at a concentration of from about 20 μM to about 1 mM, from about30 μM to about 900 μM, from about 40 μM to about 700 μM, from about 50μM to about 500 μM, from about 50 μM to about 300 μM, or from about 60μM to about 100 μM. NAD is present in the assay mixture at aconcentration of about 1 mM. The acetyl group on the tubulin peptide isradiolabeled, e.g., ¹⁴C-acetyl is used. The assay then involvesdetermining the amount of ¹⁴C-acetyl that is released, typically byscintillation counting.

Another method of detecting tubulin deacetylase activity is to monitorthe acetylation status of tubulin using an antibody specific foracetylated tubulin. Lack of reactivity of the anti-acetylated tubulinantibody with the tubulin substrate indicates that the tubulin has beendeacetylated. An example of such an antibody is the 6-11B-1 antibody, asdescribed in the Examples. Thus, in some embodiments, the methodsinvolve determining binding of an anti-acetylated tubulin antibody withthe tubulin substrate. Anti-acetylated antibody/tubulin binding can bedetermined using any type of immunological assay, includingimmunoblotting assays, ELISA assays, and the like.

In some embodiments, the assay is a cell-free assay, wherein the tubulindeacetylase is contacted with the test agent, the substrate (i.e.,acetylated tubulin), and other reaction components (e.g., NAD, buffers,and the like), and the activity of the tubulin deacetylase determined.In these embodiments, the tubulin deacetylase may be purified, but neednot be. The tubulin deacetylase may be present in a cell extract; in animmunoprecipitate of a cell extract; or may be partially purified, e.g.,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or more, purified, e.g., free of othermacromolecules present in the source of the tubulin deacetylase. Thetubulin deacetylase may be recombinant, or may be isolated from anatural source, e.g., a mammalian cell or tissue that normally producedthe enzyme.

In other embodiments, the assay is a cell-based in vitro assay, whereinthe cell is contacted with the test agent, and the effect, if any, ofthe agent on the activity of tubulin deacetylase is determined. In theseembodiments, the effect of the agent on tubulin deacetylase enzymaticactivity is determined by monitoring the acetylation status of tubulinin the cell. The methods involve contacting the cell with the testagent, and, after a suitable period of time, tubulin is extracted fromthe cell, and the degree of acetylation is determined. The degree ofacetylation of tubulin can be determined using any known method,including, e.g., binding of an anti-acetylated tubulin antibody to thetubulin extracted from the cell.

In some embodiments, the assay method involves determining MIZ-1 bindingto acetylated tubulin. MIZ-1/tubulin binding can be measured using anyknown assay, including well-known protein-protein binding assays.Suitable methods include: a yeast two-hybrid method; a fluorescenceresonance energy transfer (FRET) assay; a bioluminenscence resonanceenergy transfer (BRET) assay; a fluorescence quenching assay; afluorescence anisotropy assay; an immunological assay; and an assayinvolving binding of a detectably labeled protein to an immobilizedprotein.

In any assay involving MIZ-1 binding to acetylated tubulin, anacetylated tubulin fragment can be used. Acetylated tubulin fragmentsare discussed above, and include, but are not limited to, a fragmentsuch as that set forth in SEQ ID NO:08. In any assay involving MIZ-1binding to acetylated tubulin, MIZ-1 as set forth in SEQ ID NO:09, or anacetylated tubulin-binding fragment of MIZ-1, can be used. In someembodiments, one or both of acetylated tubulin and MIZ-1 protein isdetectably labeled.

FRET involves the transfer of energy from a donor fluorophore in anexcited state to a nearby acceptor fluorophore. For this transfer totake place, the donor and acceptor molecules must in close proximity(e.g., less than 10 nanometers apart, usually between 10 and 100 Åapart), and the emission spectra of the donor fluorophore must overlapthe excitation spectra of the acceptor fluorophore.

In these embodiments, a fluorescently labeled MIZ-1 protein or a tubulinprotein serves as a donor and/or acceptor in combination with a secondfluorescent protein or dye, e.g., a fluorescent protein as described inMatz et al., Nature Biotechnology (October 1999) 17:969-973; a greenfluorescent protein (GFP), including a “humanized” GFP; a GFP fromAequoria Victoria or fluorescent mutant thereof, e.g., as described inU.S. Pat. Nos. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750;5,968,738; 5,958,713; 5,919,445; 5,874,304, the disclosures of which areherein incorporated by reference; a GFP from another species such asRenilla reniformis, Renilla mulleri, or Ptilosarcus guernyi, asdescribed in, e.g., WO 99/49019 and Peelle et al. (2001) J. ProteinChem. 20:507-519; “humanized” recombinant GFP (hrGFP) (Stratagene);other fluorescent dyes, e.g., coumarin and its derivatives, e.g.7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes, such as BodipyFla., cascade blue, fluorescein and its derivatives, e.g. fluoresceinisothiocyanate, Oregon green, rhodamine dyes, e.g. texas red,tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g. Cy3 andCy5, macrocyclic chelates of lanthanide ions, e.g. quantum dye, etc.,chemilumescent dyes, e.g., luciferases.

BRET is a protein-protein interaction assay based on energy transferfrom a bioluminescent donor to a fluorescent acceptor protein. The BRETsignal is measured by the amount of light emitted by the acceptor to theamount of light emitted by the donor. The ratio of these two valuesincreases as the two proteins are brought into proximity. The BRET assayhas been amply described in the literature. See, e.g., U.S. Pat. Nos.6,020,192; 5,968,750; and 5,874,304; and Xu et al. (1999) Proc. Natl.Acad. Sci. USA 96:151-156. BRET assays may be performed by analyzingtransfer between a bioluminescent donor protein and a fluorescentacceptor protein. Interaction between the donor and acceptor proteinscan be monitored by a change in the ratio of light emitted by thebioluminescent and fluorescent proteins. In this application, the MIZ-1protein or the tubulin protein serves as donor and/or acceptor protein.

Agents

The present invention further provides biologically active agentsidentified using a method of the instant invention. A biologicallyactive agent of the invention modulates a level or an activity of atubulin deacetylase. In some embodiments, an agent that inhibits atubulin deacetylase is useful in a method of stabilizing tubulin,thereby reducing cell proliferation, and thus is useful to treat cancer.

In many embodiments, the agent is a small molecule, e.g., a smallorganic or inorganic compound having a molecular weight of more than 50and less than about 2,500 daltons. Agents may comprise functional groupsnecessary for structural interaction with proteins, particularlyhydrogen bonding, and may include at least an amine, carbonyl, hydroxylor carboxyl group, and may contain at least two of the functionalchemical groups. The agents may comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

In some embodiments, an active agent is a peptide. Suitable peptidesinclude peptides of from about 3 amino acids to about 50, from about 5to about 30, or from about 10 to about 25 amino acids in length. Apeptide of interest inhibits an enzymatic activity of tubulindeacetylase.

Peptides can include naturally-occurring and non-naturally occurringamino acids. Peptides may comprise D-amino acids, a combination of D-and L-amino acids, and various “designer” amino acids (e.g., β-methylamino acids, Cα-methyl amino acids, and Nα-methyl amino acids, etc.) toconvey special properties to peptides. Additionally, peptide may be acyclic peptide. Peptides may include non-classical amino acids in orderto introduce particular conformational motifs. Any known non-classicalamino acid can be used. Non-classical amino acids include, but are notlimited to, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate;(2S,3S)-methylphenylalanine, (2S,3R)-methyl-phenylalanine,(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine;2-aminotetrahydronaphthalene-2-carboxylic acid;hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate; β-carboline (D andL); HIC (histidine isoquinoline carboxylic acid); and HIC (histidinecyclic urea). Amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures,including, but not limited to, LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog; β-sheet inducing analogs; β-turn inducing analogs;α-helix inducing analogs; γ-turn inducing analogs; Gly-Ala turn analog;amide bond isostere; tretrazol; and the like.

A peptide may be a depsipeptide, which may be a linear or a cyclicdepsipeptide. Kuisle et al. (1999) Tet. Letters 40:1203-1206.“Depsipeptides” are compounds containing a sequence of at least twoalpha-amino acids and at least one alpha-hydroxy carboxylic acid, whichare bound through at least one normal peptide link and ester links,derived from the hydroxy carboxylic acids, where “linear depsipeptides”may comprise rings formed through S—S bridges, or through an hydroxy ora mercapto group of an hydroxy-, or mercapto-amino acid and the carboxylgroup of another amino- or hydroxy-acid but do not comprise rings formedonly through peptide or ester links derived from hydroxy carboxylicacids. “Cyclic depsipeptides” are peptides containing at least one ringformed only through peptide or ester links, derived from hydroxycarboxylic acids.

Peptides may be cyclic or bicyclic. For example, the C-terminal carboxylgroup or a C-terminal ester can be induced to cyclize by internaldisplacement of the —OH or the ester (—OR) of the carboxyl group orester respectively with the N-terminal amino group to form a cyclicpeptide. For example, after synthesis and cleavage to give the peptideacid, the free acid is converted to an activated ester by an appropriatecarboxyl group activator such as dicyclohexylcarbodiimide (DCC) insolution, for example, in methylene chloride (CH₂Cl₂), dimethylformamide (DMF) mixtures. The cyclic peptide is then formed by internaldisplacement of the activated ester with the N-terminal amine. Internalcyclization as opposed to polymerization can be enhanced by use of verydilute solutions. Methods for making cyclic peptides are well known inthe art

The term “bicyclic” refers to a peptide in which there exists two ringclosures. The ring closures are formed by covalent linkages betweenamino acids in the peptide. A covalent linkage between two nonadjacentamino acids constitutes a ring closure, as does a second covalentlinkage between a pair of adjacent amino acids which are already linkedby a covalent peptide linkage. The covalent linkages forming the ringclosures may be amide linkages, i.e., the linkage formed between a freeamino on one amino acid and a free carboxyl of a second amino acid, orlinkages formed between the side chains or “R” groups of amino acids inthe peptides. Thus, bicyclic peptides may be “true” bicyclic peptides,i.e., peptides cyclized by the formation of a peptide bond between theN-terminus and the C-terminus of the peptide, or they may be“depsi-bicyclic” peptides, i.e., peptides in which the terminal aminoacids are covalently linked through their side chain moieties.

A desamino or descarboxy residue can be incorporated at the terminii ofthe peptide, so that there is no terminal amino or carboxyl group, todecrease susceptibility to proteases or to restrict the conformation ofthe peptide. C-terminal functional groups include amide, amide loweralkyl, amide di(lower alkyl), lower alkoxy, hydroxy, and carboxy, andthe lower ester derivatives thereof, and the pharmaceutically acceptablesalts thereof.

In addition to the foregoing N-terminal and C-terminal modifications, apeptide or peptidomimetic can be modified with or covalently coupled toone or more of a variety of hydrophilic polymers to increase solubilityand circulation half-life of the peptide. Suitable nonproteinaceoushydrophilic polymers for coupling to a peptide include, but are notlimited to, polyalkylethers as exemplified by polyethylene glycol andpolypropylene glycol, polylactic acid, polyglycolic acid,polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose andcellulose derivatives, dextran and dextran derivatives, etc. Generally,such hydrophilic polymers have an average molecular weight ranging fromabout 500 to about 100,000 daltons, from about 2,000 to about 40,000daltons, or from about 5,000 to about 20,000 daltons. The peptide can bederivatized with or coupled to such polymers using any of the methodsset forth in Zallipsky, S., Bioconjugate Chem., 6:150-165 (1995);Monfardini, C, et al., Bioconjugate Chem., 6:62-69 (1995); U.S. Pat.Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; 4,179,337 orWO 95/34326.

Another suitable agent for reducing an activity of a tubulin deacetylaseis a peptide aptamer. Peptide aptamers are peptides or smallpolypeptides that act as dominant inhibitors of protein function.Peptide aptamers specifically bind to target proteins, blocking theirfunction ability. Kolonin and Finley, PNAS (1998) 95:14266-14271. Due tothe highly selective nature of peptide aptamers, they may be used notonly to target a specific protein, but also to target specific functionsof a given protein (e.g. a signaling function). Further, peptideaptamers may be expressed in a controlled fashion by use of promoterswhich regulate expression in a temporal, spatial or inducible manner.Peptide aptamers act dominantly; therefore, they can be used to analyzeproteins for which loss-of-function mutants are not available.

Peptide aptamers that bind with high affinity and specificity to atarget protein may be isolated by a variety of techniques known in theart. Peptide aptamers can be isolated from random peptide libraries byyeast two-hybrid screens (Xu et al., PNAS (1997) 94:12473-12478). Theycan also be isolated from phage libraries (Hoogenboom et al.,Immunotechnology (1998) 4:1-20) or chemically generatedpeptides/libraries.

Intracellularly expressed antibodies, or intrabodies, are single-chainantibody molecules designed to specifically bind and inactivate targetmolecules inside cells. Intrabodies have been used in cell assays and inwhole organisms. Chen et al., Hum. Gen. Ther. (1994) 5:595-601;Hassanzadeh et al., Febs Lett. (1998) 16(1, 2):75-80 and 81-86.Inducible expression vectors can be constructed with intrabodies thatreact specifically with tubulin deacetylase protein. These vectors canbe introduced into model organisms and studied in the same manner asdescribed above for aptamers.

In some of the invention, the active agent is an agent that modulates,and generally decreases or down regulates, the expression of the geneencoding tubulin deacetylase in the host. Such agents include, but arenot limited to, antisense RNA, interfering RNA, ribozymes, and the like.

In some embodiments, the active agent is an interfering RNA (RNAi). RNAiincludes double-stranded RNA interference (dsRNAi). Use of RNAi toreduce a level of a particular mRNA and/or protein is based on theinterfering properties of double-stranded RNA derived from the codingregions of gene. In one example of this method, complementary sense andantisense RNAs derived from a substantial portion of the tubulindeacetylase gene are synthesized in vitro. The resulting sense andantisense RNAs are annealed in an injection buffer, and thedouble-stranded RNA injected or otherwise introduced into the subject(such as in their food or by soaking in the buffer containing the RNA).See, e.g., WO99/32619. In another embodiment, dsRNA derived from atubulin deacetylase gene is generated in vivo by simultaneous expressionof both sense and antisense RNA from appropriately positioned promotersoperably linked to tubulin deacetylase coding sequences in both senseand antisense orientations.

Antisense molecules can be used to down-regulate expression of the geneencoding tubulin deacetylase in cells. Antisense compounds includeribozymes, external guide sequence (EGS) oligonucleotides (oligozymes),and other short catalytic RNAs or catalytic oligonucleotides whichhybridize to the target nucleic acid and modulate its expression.

The anti-sense reagent may be antisense oligonucleotides (ODN),particularly synthetic ODN having chemical modifications from nativenucleic acids, or nucleic acid constructs that express such anti-sensemolecules as RNA. The antisense sequence is complementary to the mRNA ofthe targeted gene, and inhibits expression of the targeted geneproducts. Antisense molecules inhibit gene expression through variousmechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part ofthe target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996), Nature Biotechnol. 14:840-844). Because the nucleotidesequence of the gene encoding human tubulin deacetylase is known (see,e.g., GenBank Accession No. NT_(—)011109; and GenBank Accession Nos.NM_(—)030593, NM_(—)012237, AJ505014, and AF083107), those skilled inthe art can readily generate antisense nucleic acids that reduce thelevel of a human tubulin deacetylase gene product in a cell.

A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in an in vitro or animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methodsknown in the art (see Wagner et al. (1993), supra, and Milligan et al.,supra.) Preferred oligonucleotides are chemically modified from thenative phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which modificationsalter the chemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates;phosphorodithioates, where both of the non-bridging oxygens aresubstituted with sulfur; phosphoroamidites; alkyl phosphotriesters andboranophosphates. Achiral phosphate derivatives include3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH₂-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The β-anomer of deoxyribose may be used, where the base isinverted with respect to the natural α-anomer. The 2′-OH of the ribosesugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars, whichprovides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

Exemplary modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Oligonucleotides having a morpholino backbone structure (Summerton, J.E. and Weller D. D., U.S. Pat. No. 5,034,506) or a peptide nucleic acid(PNA) backbone (P. E. Nielson, M. Egholm, R. H. Berg, O. Buchardt,Science 1991, 254: 1497) can also be used. Morpholino antisenseoligonucleotides are amply described in the literature. See, e.g.,Partridge et al. (1996) Antisense Nucl. Acid Drug Dev. 6:169-175; andSummerton (1999) Biochem. Biophys. Acta 1489:141-158.

As an alternative to anti-sense inhibitors, catalytic nucleic acidcompounds, e.g. ribozymes, anti-sense conjugates, etc. may be used toinhibit gene expression. Ribozymes may be synthesized in vitro andadministered to the patient, or may be encoded on an expression vector,from which the ribozyme is synthesized in the targeted cell (forexample, see International patent application WO 9523225, and Beigelmanet al. (1995), Nucl. Acids Res. 23:4434-42). Examples ofoligonucleotides with catalytic activity are described in WO 9506764.Conjugates of anti-sense ODN with a metal complex, e.g.terpyridylCu(II), capable of mediating mRNA hydrolysis are described inBashkin et al. (1995), Appl. Biochem. Biotechnol. 54:43-56.

Formulations, Dosages, and Routes of Administration

The invention provides formulations, including pharmaceuticalformulations, comprising an agent that reduces a level and/or anactivity of tubulin deacetylase. In general, a formulation comprises aneffective amount of an agent that reduces a level and/or an activity oftubulin deacetylase. An “effective amount” means a dosage sufficient toproduce a desired result, e.g., a reduction in a level and/or anactivity of tubulin deacetylase, stabilization of microtubules; areduction in tubulin deacetylation; a reduction in cell proliferation;and the like. Generally, the desired result is at least a reduction alevel and/or an activity of tubulin deacetylase as compared to acontrol.

Formulations

In the subject methods, the active agent(s) may be administered to thehost using any convenient means capable of resulting in the desiredreduction in a level and/or an activity of tubulin deacetylase. Thus,the agent can be incorporated into a variety of formulations fortherapeutic administration. More particularly, the agents of the presentinvention can be formulated into pharmaceutical compositions bycombination with appropriate, pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.

In pharmaceutical dosage forms, the agents may be administered in theform of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds. The following methods andexcipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The agents can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation. The compounds of the present invention can be formulatedinto pressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Thecompounds of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or moreinhibitors. Similarly, unit dosage forms for injection or intravenousadministration may comprise the inhibitor(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

Other modes of administration will also find use with the subjectinvention. For instance, an agent of the invention can be formulated insuppositories and, in some cases, aerosol and intranasal compositions.For suppositories, the vehicle composition will include traditionalbinders and carriers such as, polyalkylene glycols, or triglycerides.Such suppositories may be formed from mixtures containing the activeingredient in the range of about 0.5% to about 10% (w/w), preferablyabout 1% to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed with the subject invention. The nasalformulations may also contain preservatives such as, but not limited to,chlorobutanol and benzalkonium chloride. A surfactant may be present toenhance absorption of the subject proteins by the nasal mucosa.

An agent of the invention can be administered as injectables. Typically,injectable compositions are prepared as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. The preparation may also beemulsified or the active ingredient encapsulated in liposome vehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of the agent adequate to achieve thedesired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

Dosages

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is one which provides up to about 1 μgto about 1,000 μg or about 10,000 μg of an agent that reduces a leveland/or an activity of tubulin deacetylase can be administered in asingle dose. Alternatively, a target dosage of an agent that reduces alevel and/or an activity of tubulin deacetylase can be considered to beabout in the range of about 0.1-1000 μM, about 0.5-500 μM, about 1-100μM, or about 5-50 μM in a sample of host blood drawn within the first24-48 hours after administration of the agent.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific compound, the severity of the symptoms and thesusceptibility of the subject to side effects. Preferred dosages for agiven compound are readily determinable by those of skill in the art bya variety of means.

Routes of Administration

An agent that reduces a level and/or an activity of tubulin deacetylaseis administered to an individual using any available method and routesuitable for drug delivery, including in vivo and ex vivo methods, aswell as systemic and localized routes of administration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, intratumoral,subcutaneous, intradermal, topical application, intravenous, rectal,nasal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe agent and/or the desired effect. The composition can be administeredin a single dose or in multiple doses.

The agent can be administered to a host using any available conventionalmethods and routes suitable for delivery of conventional drugs,including systemic or localized routes. In general, routes ofadministration contemplated by the invention include, but are notnecessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, and intravenous routes, i.e., any route of administrationother than through the alimentary canal. Parenteral administration canbe carried to effect systemic or local delivery of the agent. Wheresystemic delivery is desired, administration typically involves invasiveor systemically absorbed topical or mucosal administration ofpharmaceutical preparations.

The agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Methods of administration of the agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

By treatment is meant at least an amelioration of the symptomsassociated with the pathological condition afflicting the host, whereamelioration is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, e.g. symptom, associated with thepathological condition being treated, such as an allergichypersensitivity. As such, treatment also includes situations where thepathological condition, or at least symptoms associated therewith, arecompletely inhibited, e.g. prevented from happening, or stopped, e.g.terminated, such that the host no longer suffers from the pathologicalcondition, or at least the symptoms that characterize the pathologicalcondition.

A variety of hosts (wherein the term “host” is used interchangeablyherein with the terms “subject” and “patient”) are treatable accordingto the subject methods. Generally such hosts are “mammals” or“mammalian,” where these terms are used broadly to describe organismswhich are within the class mammalia, including the orders carnivore(e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), andprimates (e.g., humans, chimpanzees, and monkeys). In many embodiments,the hosts will be humans.

Kits with unit doses of the active agent, e.g. in oral or injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of the drugs in treatingpathological condition of interest. Preferred compounds and unit dosesare those described herein above.

Combination Therapies

The present invention also provides methods of treating cancer, andmethods of reducing unwanted cellular proliferation, involvingadministering an agent that modulates (e.g., inhibits) a tubulindeacetylase; and a second therapeutic agent. In some embodiments, anagent that inhibits a tubulin deacetylase is administered as an adjuvantto a standard cancer therapy.

Standard cancer therapies include surgery (e.g., surgical removal ofcancerous tissue), radiation therapy, bone marrow transplantation,chemotherapeutic treatment, biological response modifier treatment, andcertain combinations of the foregoing.

Radiation therapy includes, but is not limited to, x-rays or gamma raysthat are delivered from either an externally applied source such as abeam, or by implantation of small radioactive sources.

Chemotherapeutic agents are non-peptidic (i.e., non-proteinaceous)compounds that reduce proliferation of cancer cells, and encompasscytotoxic agents and cytostatic agents. Non-limiting examples ofchemotherapeutic agents include alkylating agents, nitrosoureas,antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, andsteroid hormones.

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatine, and gemcitabine.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel(Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide, etc.;antibiotics, e.g. anthracycline, daunorubicin hydrochloride (daunomycin,rubidomycin, cerubidine), idarubicin, doxorubicin, epirubicin andmorpholino derivatives, etc.; phenoxizone biscyclopeptides, e.g.dactinomycin; basic glycopeptides, e.g. bleomycin; anthraquinoneglycosides, e.g. plicamycin (mithramycin); anthracenediones, e.g.mitoxantrone; azirinopyrrolo indolediones, e.g. mitomycin; macrocyclicimmunosuppressants, e.g. cyclosporine, FK-506 (tacrolimus, prograf),rapamycin, etc.; and the like.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,ifosamide, and droloxafine.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®.Estrogens stimulate proliferation and differentiation, thereforecompounds that bind to the estrogen receptor are used to block thisactivity. Corticosteroids may inhibit T cell proliferation.

Other chemotherapeutic agents include metal complexes, e.g. cisplatin(cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines,e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor;procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

“Taxanes” include paclitaxel, as well as any active taxane derivative orpro-drug. “Paclitaxel” (which should be understood herein to includeanalogues, formulations, and derivatives such as, for example,docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), 10-desacetylanalogs of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs ofpaclitaxel) may be readily prepared utilizing techniques known to thoseskilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253;5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267),or obtained from a variety of commercial sources, including for example,Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; orT-1912 from Taxus yannanensis).

Paclitaxel should be understood to refer to not only the commonchemically available form of paclitaxel, but analogs and derivatives(e.g., Taxotere™ docetaxel, as noted above) and paclitaxel conjugates(e.g., paclitaxel-PEG, paclitaxel-dextran, or paclitaxel-xylose).

Also included within the term “taxane” are a variety of knownderivatives, including both hydrophilic derivatives, and hydrophobicderivatives. Taxane derivatives include, but not limited to, galactoseand mannose derivatives described in International Patent ApplicationNo. WO 99/18113; piperazino and other derivatives described in WO99/14209; taxane derivatives described in WO 99/09021, WO 98/22451, andU.S. Pat. No. 5,869,680; 6-thio derivatives described in WO 98/28288;sulfenamide derivatives described in U.S. Pat. No. 5,821,263; and taxolderivative described in U.S. Pat. No. 5,415,869. It further includesprodrugs of paclitaxel including, but not limited to, those described inWO 98/58927; WO 98/13059; and U.S. Pat. No. 5,824,701.

Biological response modifiers suitable for use in connection with themethods of the invention include, but are not limited to, (1) inhibitorsof tyrosine kinase (RTK) activity; (2) inhibitors of serine/threoninekinase activity; (3) tumor-associated antigen antagonists, such asantibodies that bind specifically to a tumor antigen; (4) apoptosisreceptor agonists; (5) interleukin-2; (6) IFN-α; (7) IFN-γ (8)colony-stimulating factors; (9) inhibitors of angiogenesis; and (10)antagonists of tumor necrosis factor.

Therapeutic Methods

The present invention provides methods of modulating tubulinacetylation; methods of stabilizing microtubules; methods of reducingunwanted cellular proliferation; and methods of treating disordersresulting from unwanted cellular proliferation. The methods generallyinvolve administering to an individual an effective amount of a subjectagent that modulates (e.g., inhibits) an enzymatic activity of tubulindeacetylase (e.g., SIRT2), in an amount effective to reduce unwantedcellular proliferation. An effective amount of a subject agent reducescell proliferation, and/or decreases tumor mass.

An agent that inhibits tubulin deacetylase enzymatic activity isadministered to a patient in need thereof, e.g., a patient who hascancer.

In the context of reducing unwanted cell proliferation, and reducingtumor mass, an effective amount of an agent that inhibits tubulindeactetylase is an amount that reduces the level and/or rate of cellproliferation and/or reduces tumor mass by at least about 10%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 80%, at least about 85%, or at least about 90%, or more,compared to the level and/or rate of cell proliferation and/or tumormass in the absence of treatment with an agent that inhibits tubulindeacetylase.

Whether a particular agent reduces the rate and/or level of cellproliferation can be determined using any known assay. For example, anin vitro assay can be used, in which cells (e.g., tumor cells) arecultured in culture medium to which has been added an agent. Cellproliferation is determined using any known assay, e.g., ³H-thymidineincorporation; counting viable cell number. Viable cell number can becounted using any known method. For example, a fluorescence activatedcell sorting (FACS) method is used to determine the number of cells thatare stained with a viable cell stain (e.g., fluorescein di-O-acetate,and the like), compared to the number of cells stained with a dye thatdoes not normally stain viable cells, such as propidium iodide.

Whether a particular therapeutic regimen of the invention is effectivein reducing unwanted cellular proliferation, e.g., in the context oftreating cancer, can be determined using standard methods. For example,the number of cancer cells in a biological sample (e.g., blood, a biopsysample, and the like), can be determined. The tumor mass can bedetermined using standard radiological methods.

Whether a tumor load has been decreased can be determined using anyknown method, including, but not limited to, measuring solid tumor mass;counting the number of tumor cells using cytological assays;fluorescence-activated cell sorting (e.g., using antibody specific for atumor-associated antigen) to determine the number of cells bearing agiven tumor antigen; computed tomography scanning, magnetic resonanceimaging, and/or x-ray imaging of the tumor to estimate and/or monitortumor size; measuring the amount of tumor-associated antigen in abiological sample, e.g., blood, serum, etc.; and the like.

Whether growth of a tumor is inhibited can be determined using any knownmethod, including, but not limited to, an in vitro cell proliferationassay (e.g., counting cell number); a ³H-thymidine uptake assay; and thelike.

An agent that inhibits tubulin deacetylase is administered by any routeof administration. Conventional and pharmaceutically acceptable routesof administration include intranasal, intramuscular, intratracheal,intratumoral, subcutaneous, intradermal, topical application,intravenous, rectal, nasal, oral and other parenteral routes ofadministration. Routes of administration may be combined, if desired, oradjusted depending upon the agent and/or the desired effect.

The agent can be administered in a single dose or in multiple doses. Forexample, an agent that inhibits tubulin deacetylase is administered onceper month, twice per month, three times per month, every other week(qow), once per week (qw), twice per week (biw), three times per week(tiw), four times per week, five times per week, six times per week,every other day (qod), daily (qd), twice a day (qid), or three times aday (tid), substantially continuously, or continuously, over a period oftime ranging from about one day to about one week, from about two weeksto about four weeks, from about one month to about two months, fromabout two months to about four months, from about four months to aboutsix months, from about six months to about eight months, from abouteight months to about 1 year, from about 1 year to about 2 years, orfrom about 2 years to about 4 years, or more.

In some embodiments, an agent that inhibits a tubulin deacetylase isadministered as an adjuvant to a standard cancer therapy, as describedabove.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is an alkylating agent. In someembodiments, the alkylating agent is a nitrogen mustard. In otherembodiments, the alkylating agent is an ethylenimine. In still otherembodiments, the alkylating agent is an alkylsulfonate. In additionalembodiments, the alkylating agent is a triazene. In further embodiments,the allylating agent is a nitrosourea.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is an antimetabolite. In someembodiments, the antimetabolite is a folic acid analog, such asmethotrexate. In other embodiments, the antimetabolite is a purineanalog, such as mercaptopurine, thioguanine and axathioprine. In stillother embodiments, the antimetabolite is a pyrimidine analog, such as5FU, UFT, capecitabine, gemcitabine and cytarabine.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is a vinca alkyloid. In someembodiments, the vinca alkaloid is a taxane, such as paclitaxel. Inother embodiments, the vinca alkaloid is a podophyllotoxin, such asetoposide, teniposide, ironotecan, and topotecan.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is an antineoplastic antibiotic.In some embodiments, the antineoplastic antibiotic is doxorubicin.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is a platinum complex. In someembodiments, the platinum complex is cisplatin. In other embodiments,the platinum complex is carboplatin.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is a tyrosine kinase inhibitor.In some embodiments, the tyrosine kinase inhibitor is a receptortyrosine kinase (RTK) inhibitor, such as type I receptor tyrosine kinaseinhibitors (e.g., inhibitors of epidermal growth factor receptors), typeII receptor tyrosine kinase inhibitors (e.g., inhibitors of insulinreceptor), type III receptor tyrosine kinase inhibitors (e.g.,inhibitors of platelet-derived growth factor receptor), and type IVreceptor tyrosine kinase inhibitors (e.g., fibroblast growth factorreceptor). In other embodiments, the tyrosine kinase inhibitor is anon-receptor tyrosine kinase inhibitor, such as inhibitors of srckinases or janus kinases.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is an inhibitor of a receptortyrosine kinase involved in growth factor signaling pathway(s). In someembodiments, the inhibitor is genistein. In other embodiments, theinhibitor is an epidermal growth factor receptor (EGFR) tyrosinekinase-specific antagonist, such as IRESSA™ gefitinib, TARCEVA™erolotinib, or tyrphostin AG1478(4-(3-chloroanilino)-6,7-dimethoxyquinazoline. In still otherembodiments, the inhibitor is any indolinone antagonist of Flk-1/KDR(VEGF-R2) tyrosine kinase activity. In further embodiments, theinhibitor is any of the substituted3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1,3-dihydroindol-2-oneantagonist of Flk-1/KDR (VEGF-R2), FGF-R1 or PDGF-R tyrosine kinaseactivity. In additional embodiments, the inhibitor is any substituted3-[(3- or 4-carboxyethylpyrrol-2-yl)methylidenyl]indolin-2-oneantagonist of Flt-1 (VEGF-R1), Flk-1/KDR (VEGF-R2), FGF-R1 or PDGF-Rtyrosine kinase activity.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is an inhibitor of anon-receptor tyrosine kinase involved in growth factor signalingpathway(s). In some embodiments, the inhibitor is an antagonist of JAK2tyrosine kinase activity, such as tyrphostin AG490(2-cyano-3-(3,4-dihydroxyphenyl)-N-(benzyl)-2-propenamide). In otherembodiments, the inhibitor is an antagonist of bcr-abl tyrosine kinaseactivity, such as GLEEVEC™ imatinib mesylate.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is a serine/threonine kinaseinhibitor. In some embodiments, the serine/threonine kinase inhibitor isa receptor serine/threonine kinase inhibitor, such as antagonists ofTGF-β receptor serine/threonine kinase activity. In other embodiments,the serine/threonine kinase inhibitor is a non-receptor serine/threoninekinase inhibitor, such as antagonists of the serine/threonine kinaseactivity of the MAP kinases, protein kinase C(PKC), protein kinase A(PKA), or the cyclin-dependent kinases (CDKs).

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the cancer patient an effective amount of at leastone additional antineoplastic drug that is an inhibitor of one or morekinases involved in cell cycle regulation. In some embodiments, theinhibitor is an antagonist of CDK2 activation, such as tryphostin AG490(2-cyano-3-(3,4-dihydroxyphenyl)-N-(benzyl)-2-propenamide). In otherembodiments, the inhibitor is an antagonist of CDK1/cyclin B activity,such as alsterpaullone. In still other embodiments, the inhibitor is anantagonist of CDK2 kinase activity, such as indirubin-3′-monoxime.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the patient an effective amount of a taxane. In someembodiments, the methods involve administering an effective amount of anagent that inhibits tubulin deacetylase, and co-administering to thepatient an effective amount of a taxane, and an effective amount of aplatinum complex. In some embodiments, the taxane is paclitaxel and theplatinum complex is cisplatin or carboplatin.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the patient an effective amount of at least oneadditional antineoplastic drug that is an a tumor-associated antigenantagonist, such as an antibody antagonist. In some embodimentsinvolving the treatment of HER2-expressing tumors, the tumor-associatedantigen antagonist is an anti-HER2 monoclonal antibody, such asHERCEPTIN™ trastuzumab. In some embodiments involving the treatment ofCD20-expressing tumors, such as B-cell lymphomas, the tumor-associatedantigen antagonist is an anti-CD20 monoclonal antibody, such as RITUXAN™rituximab.

In some embodiments, the methods involve administering an effectiveamount of an agent that inhibits tubulin deacetylase, andco-administering to the patient an effective amount of at least oneadditional antineoplastic drug that is a tumor growth factor antagonist.In some embodiments, the tumor growth factor antagonist is an antagonistof epidermal growth factor (EGF), such as an anti-EGF monoclonalantibody. In other embodiments, the tumor growth factor antagonist is anantagonist of epidermal growth factor receptor erbB1 (EGFR), such as ananti-EGFR monoclonal antibody antagonist of EGFR activation or signaltransduction.

In some embodiments, the agent that inhibits tubulin deacetylase isribavirin or a ribavirin derivative. Ribavirin,1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available from ICNPharmaceuticals, Inc., Costa Mesa, Calif., is described in the MerckIndex, compound No. 8199, Eleventh Edition. Its manufacture andformulation is described in U.S. Pat. No. 4,211,771. The invention alsocontemplates use of derivatives of ribavirin (see, e.g., U.S. Pat. No.6,277,830). The ribavirin may be administered orally in capsule ortablet form. Of course, other types of administration, as they becomeavailable are contemplated, such as by nasal spray, transdermally,intravenously, by suppository, by sustained release dosage form, etc.Any form of administration will work so long as the proper dosages aredelivered without destroying the active ingredient.

Ribavirin is generally administered in an amount ranging from about 30mg to about 1200 mg per day, e.g., from about 30 mg to about 60 mg, fromabout 60 mg to about 125 mg, from about 125 mg to about 200 mg, fromabout 200 mg to about 300 mg, from about 300 mg to about 400 mg, fromabout 400 mg to about 600 mg, from about 600 mg to about 800 mg, fromabout 800 mg to about 1000 mg, or from about 1000 mg to about 1200 mgper day.

Exemplary non-limiting examples of combination therapies that includetreatment with radiation, tubulin deacetylase inhibiting agent, ortreatment with a chemotherapeutic agent and tubulin deacetylaseinhibiting agent, are as follows:

1) a dosage of an agent that inhibits tubulin deacetylase; and cisplatinin a dosage range of from about 5 mg/m² to about 150 mg/m²;

2) a dosage of an agent that inhibits tubulin deacetylase; andcarboplatin in a dosage range of from about 5 mg/m² to about 1000 mg/m²;

3) a dosage of an agent that inhibits tubulin deacetylase; and radiationin a dosage range of from about 200 cGy to about 8000 cGy;

4) a dosage of an agent that inhibits tubulin deacetylase; andpaclitaxel in a dosage range of from about 40 mg/m² to about 250 mg/m²;

5) a dosage of an agent that inhibits tubulin deacetylase; paclitaxel ina dosage range of from about 40 mg/m² to about 250 mg/m²; andcarboplatin in a dosage range of from about 5 mg/m² to about 1000 mg/m²;

6) a dosage of an agent that inhibits tubulin deacetylase; 5FU in adosage range of from about 5 mg/m² to about 5000 mg/m²; and leucovorinin a dosage range of from about 5 mg/m² to about 1000 mg/m²;

7) a dosage of an agent that inhibits tubulin deacetylase; andtrastuzumab in an initial loading dose of 4 mg/kg and a weeklymaintenance dose of 2 mg/kg;

8) a dosage of an agent that inhibits tubulin deacetylase; trastuzumabin an initial loading dose of 4 mg/kg and a weekly maintenance dose of 2mg/kg; and paclitaxel in a dosage range of from about 40 mg/m² to about250 mg/m²;

9) a dosage of an agent that inhibits tubulin deacetylase; paclitaxel ina dosage range of from about 40 mg/m² to about 250 mg/m²; andestramustine phosphate (Emcyte®) in a dosage range of from about 5 mg/m²to about 1000 mg/m²;

10) a dosage of an agent that inhibits tubulin deacetylase; cisplatin ina dosage range of from about 5 mg/m² to about 150 mg/m²; and 5FU in adosage range of from about 5 mg/m² to about 5000 mg/m².

11) dosage of an agent that inhibits tubulin deacetylase; 5FU in adosage range of from about 5 mg/m² to about 5000 mg/m²; and radiation ina dose of from about 200 cGy to about 8000 cGy; and

12) dosage of an agent that inhibits tubulin deacetylase; 5FU in adosage range of from about 5 mg/m² to about 5000 mg/m²; and paclitaxelin a dosage range of from about 40 mg/m² to about 250 mg/m².

In any of examples 1-12 of combination therapies discussed above,ribavirin in a dose of from about 30 to about 1200 mg/day can beadministered to the patient orally.

Subject Suitable for Treatment

An agent that reduces a level of a tubulin deacetylase and/or thatinhibits a tubulin deacetylase enzymatic activity is useful for treatingcancer in a patient having a cancer. The methods are useful for treatinga wide variety of cancers, including carcinomas, sarcomas, leukemias,and lymphomas. A patient having any cancer is suitable for treatmentwith a subject method.

Carcinomas that can be treated using a subject method include, but arenot limited to, esophageal carcinoma, hepatocellular carcinoma, basalcell carcinoma (a form of skin cancer), squamous cell carcinoma (varioustissues), bladder carcinoma, including transitional cell carcinoma (amalignant neoplasm of the bladder), bronchogenic carcinoma, coloncarcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma,including small cell carcinoma and non-small cell carcinoma of the lung,adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma,breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renalcell carcinoma, ductal carcinoma in situ or bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervicalcarcinoma, uterine carcinoma, testicular carcinoma, osteogeniccarcinoma, epithelieal carcinoma, and nasopharyngeal carcinoma, etc.

Sarcomas that can be treated using a subject method include, but are notlimited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma,rhabdomyosarcoma, and other soft tissue sarcomas.

Other solid tumors that can be treated using a subject method include,but are not limited to, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Leukemias that can be treated using a subject method include, but arenot limited to, a) chronic myeloproliferative syndromes (neoplasticdisorders of multipotential hematopoietic stem cells); b) acutemyelogenous leukemias (neoplastic transformation of a multipotentialhematopoietic stem cell or a hematopoietic cell of restricted lineagepotential; c) chronic lymphocytic leukemias (CLL; clonal proliferationof immunologically immature and functionally incompetent smalllymphocytes), including B-cell CLL, T-cell CLL prolymphocytic leukemia,and hairy cell leukemia; and d) acute lymphoblastic leukemias(characterized by accumulation of lymphoblasts). Lymphomas that can betreated using a subject method include, but are not limited to, B-celllymphomas (e.g., Burkitt's lymphoma); Hodgkin's lymphoma; and the like.

REFERENCES

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EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric. Standard abbreviations may beused, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s,second(s); min, minute(s); hr, hour(s); and the like.

Example 1 Characterization of Human SIRT2

Experimental Procedures

Tissue Culture

HEK 293T and HeLa were obtained from ATCC, grown in DMEM supplementedwith 10% Fetal Bovine Serum (Gemini Bio-products, Woodland, Calif.) inthe presence of penicillin, streptomycin and 2 mM L-Glutamine (GibcoInvitrogen Corp., Carlsbad, Calif.). HepG2 was obtained form ATCC andgrown in medium as described for above with the addition of 0.1 mM MEMnon-essential amino acids (Gibco Invitrogen Corp., Carlsbad, Calif.).

Plasmids and Mutagenesis

For recombinant SIRT2, human SIRT2 cDNA in pHEX (a gift from R. Frye)was altered to insert a factor Xa protease cleavage site. Human SIRT1and SIRT3 constructs were also a gift from R. Frye. Human SIRT4-7 werecloned from testis and spleen cDNA libraries (Clontech, Mountain View,Calif.) into pcDNA3.1(+) vector by standard of PCR-based strategies andconfirmed by sequencing. All SIRT cDNAs were sub-cloned to generateC-terminal FLAG-tagged fusions in the pcDNA3.1 (+) backbone (InVitrogen,Carlsbad, Calif.) and wild type human SIRT2 was cloned into pEGFP-C1vector (Clontech, Mountain View, Calif.) by standard PCR-basedstrategies. pEGFP-MIZ-1 was a kind gift from J. E. Wilson. Site directedmutagenesis for SIRT2 constructs were performed using QuikChangeSite-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) performedas described by manufacturer.

Purification of Recombinant SIRT2

DH5αFIQ bacteria (Gibco Invitrogen Corp., Carlsbad, Calif.) weretransformed with pHEX vector containing the human SIRT2 cDNA with factorXa cleavage site and induced with 0.1 mM IPTG at 37° C. for 2 h.Resultant 6× His-tagged protein was purified under native conditions at4° C. by Ni-NTA (Qiagen, Valencia, Calif.), HiPrep 26/10 Desalting andSepharose Q chromatographies (Amersham Pharmacia Biotech, Inc.,Piscataway, N.J.). Recombinant protein was aliquoted and stored at −20°C.

Transient Transfections and Immunoprecipitations

HEK 293T cells were transfected by calcium phosphate DNA precipitationmethod and lysed 48 hours post-transfection in low stringency lysisbuffer (50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 0.5% NP-40, 150 mM NaCl) inthe presence of protease inhibitor cocktail (Complete, Roche MolecularBiochemicals, Indianapolis, Ind.). FLAG tagged proteins wereimmunoprecipitated with anti-FLAG M2 agarose affinity gel (Sigma, St.Louis, Mo.) and GFP-tagged proteins were immunoprecipitated withanti-GFP monoclonal antibody (Sigma, St. Louis, Mo.) in the presence ofProtein G Sepharose (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.)for 2 hours at 4° C. Immunoprecipitated material was washed 3 times inlow stringency lysis buffer and one time in SIRT2 deacetylase buffer (50mM Tris-HCl, pH 9.0, 4 mM MgCl₂, and 0.2 mM dithiothreitol (DTT)).

Nuclear and Cytoplasmic Extracts.

HEK 293T cells were transfected as described above and subjected tonuclear and cytoplasmic extraction as described previously (Dignam etal., 1983) modified by the addition of 1.0% NP-40 to buffer C.

Histone Deacetylase Assay

Immunoprecipitated material and recombinant SIRT2 were resuspended in100 μL of SIRT2 deacetylase buffer containing NAD (Sigma, St. Louis,Mo.) and [³H] acetylated histone H4 peptide (a.a. 1-23) (Emiliani etal., 1998). TSA (WACO BioProducts, Richmond, Va.) was resuspended indimethyl sulfoxide (DMSO) was further diluted in DMSO and added toreactions to desired concentration. Reactions were incubated for 2 hoursat room temperature and stopped by the addition of 25 μL 0.1 M HCl and0.16 M acetic acid. Released acetate was extracted in 500 μL ethylacetate, and vortexed for 15 minutes. After centrifugation for 5minutes, 400 μL of the ethyl acetate fraction was mixed with 5 mlscintillation fluid and counted.

Western Blotting

Samples were separated on 10% sodium dodecyl sulfate(SDS)-polyacrylamide gels and transferred to Hybond ECL nitrocellulosemembrane (Amersham Pharmacia Biotech, Inc., Piscataway, N.J.). Membraneswere blocked with 5% blocking reagent (Bio-Rad, Hercules, Calif.) inTBS-Tween (10 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% Tween-20), theywere probed with anti-acetylated tubulin 6-11B-1, anti-tubulin B-5-1-2or anti-FLAG M2 (Sigma, St. Louis, Mo.) all at 1:2000 dilution oranti-living colors peptide antibody (Clontech, Mountain View, Calif.),anti-p65 (Santa Cruz Biotech, Santa Cruz, Calif.), or anti-Lamin A (CellSignaling Technology, Inc., Beverly, Mass.) all diluted at 1:1000.Secondary detection was performed using horseradish peroxidase-coupledsheep anti-mouse IgG (Amersham Pharmacia Biotech, Inc., Piscataway,N.J.) or goat anti-rabbit IgG (Pierce Chemical Co., Rockford, Ill.) bothdiluted 1:5000 and ECL western blotting detection system (AmershamPharmacia Biotech, Inc., Piscataway, N.J.).

Immunofluorescence

HeLa cells grown on coverslips were transfected with LipofectAMINEreagent (Gibco Invitrogen Corp., Carlsbad, Calif.) for 8 hours accordingto manufacturer's protocol or by calcium phosphate DNA precipitationmethod. Transfected cells were incubated for 12 hours with 400 nM TSA(WACO BioProducts, Richmond, Va.) 24 hours after transfection. Cellswere washed twice for 10 minutes in PBS and fixed for 10 minutes in 4%formaldehyde (EMS, Ft. Washington, Pa.) followed by permeabilization for10 minutes in 0.5% Triton-X-100 in PBS. Following three washes for 10minutes in PBS, cells were incubated for 10 minutes in 10% BSA, andincubated for 1 hour with anti-acetylated tubulin 6-11B-1, anti-tubulinB-5-1-2 diluted 1:1000, or anti-FLAG M2 diluted 1:500 in 0.1% TWEEN-20™non-ionic detergent. Cells were washed three times in 0.1% TWEEN-20™non-ionic detergent, followed by incubation with goat anti-mouse IgG (Fcspecific) TRITC-conjugated antibody (Sigma, St. Louis) diluted 1:100 in0.1% TWEEN-20™ non-ionic detergent. Following incubation cells wereincubated for 5 minutes in 20 mg/ml 4′,6′-diamidino-2-phenylindolehydrochloride (DAPI) washed three times in phosphate-buffered saline(PBS), once in ddH₂O, and mounted with Gel Mount (Biomeda Corp., FosterCity, Calif.). Slides were visualized on a Nikon E600 microscope systemequipped with a SPOT 2 Digital Camera. Confocal images were acquired bylaser-scanning confocal microscopy with an Olympus BX60 microscopeequipped with a Radiance 2000 confocal setup (BioRad, Hercules, Calif.).

In Vitro Deacetylation Assay

Immunoprecipitations were resuspended in 100 μl SIRT deacetylase buffercontaining 50 μg total cellular lysate from untransfected HEK 293T cellsand 1 mM NAD. Reactions containing 400 nM TSA or 5 mM nicotinamide(Sigma, St. Louis, Mo.) were pre-incubated at room temperature with allcomponents of the reaction in the absence of NAD for 10 minutes,Following the pre-incubation, the enzymatic reactions were started withthe addition of NAD followed by incubation for 2 hours at roomtemperature with constant agitation. Reactions were stopped by theaddition of 50 μl 6× SDS-polyacrylamide gel electrophoresis (PAGE)buffer. Beads were pelleted by centrifugation at 14,000 rpm for 10minutes and 10 μl of each supernatant was separated on 10% SDS-PAGE gelsand western blotted as described above.

Tubulin and Histone H3 Peptide Kinetics with Hst2p and SIRT2

Increasing concentrations of tubulin peptide (20-900 μM), and 800 μM NADwere reacted in the presence of 0.8 to 1 μM recombinant SIRT2 in 50 mMTris, pH 7.5, 1 mM DTT, and 10% methanol at 37° C. For Hst2p reactions,tubulin peptides and 500 μM NAD were reacted in the presence of 9 to 19μM recombinant Hst2p under the same conditions. Reactions were quenchedwith trifluoroacetic acid (TFA) to a final concentration of 1%. Timepoints were chosen such that initial velocity conditions were observed.Samples were injected into the high performance liquid chromatography(HPLC) with a Beckman C18 analytical column. Upon injection, the systemwas run isocratically with solvent A (0.05% TFA in H₂O), followed by agradient of 0-10% solvent B (0.02% TFA in CH₃CN) for 4 minutes, andfollowed by a gradient of 10-23% solvent B for 23 minutes. Deacetylatedand acetylated peptides eluted at 16% and 18% CH₃CN, respectively.Elution of substrates and products were monitored by measuring theabsorbance at 214 nM, and corresponding peaks were integrated using theBeckman System Gold Nouveau software. The amount of product wasquantified by calculating the percentage of the deacetylated tubulinpeptide from the total tubulin peptide based upon their integrationvalues. Graphs of rate versus tubulin peptide concentrations were fittedto the Michaelis-Menten equation to obtain the kinetic parameters ofK_(m), k_(cat), and V/K.

The monoacetylated histone H3 peptide ARTKQTARKSTGG(AcK)APRKQL (SEQ IDNO:03) (AcLys-14H3) was utilized as substrate for both Hst2p and SIRT2.H3 peptide was H-labeled using the histone acetyltransferase PCAF andpurified as described previously (Tanner et al., 2000). Ratemeasurements utilized a charcoal-binding assay where 70 μL of HDACreactions were quenched in 10 μL of charcoal (1:3 charcoal volume toglycine buffer volume) containing 2 M Glycine at pH 10.0. Reaction timeswere chosen (usually 2-5 minutes) such that steady-state initialvelocities were maintained. Samples were immediately heated for ≧20minutes to liberate free acetate from Ac-ADP ribose prior tocentrifugation. The supernatant was treated to an additional 10 μL ofcharcoal slurry before determining the total free acetate liberated (byliquid scintillation counting). Data were converted to initial rates andfitted to the Michaelis-Menten equation ν₀=([E]₀k_(cat)●[S]/(K_(m)+[S]).Control experiments indicated that [³H]-Ac-Lys H3 peptide was nothydrolyzed nonenzymatically. Also, addition of activated charcoal (atall pH values and temperatures examined) immediately stopped theenzymatic reaction. Heating at high pH was only necessary to liberateacetate from the Ac-ADP ribose.

Core Histones and Tubulin Proteins as Substrates

Calf thymus core histones (Calbiochem, San Diego, Calif.), wereacetylated using 7.5-10 mg/mL calf thymus histones, 100 μM AcCoA (˜150cpm/pmole, NEN), 10 μM PCAF, in [5 mM DTT, 50 mM, Tris, pH 7.5] for 1.5h at 23° C. Remaining Acetyl-CoA was removed by gel-filtration andlabeled histones were quantified by liquid scintillation counting.Deacetylation assays were performed using 30-50 nM enzyme, 500 μM NAD,˜1.2 μM histones, pH 7.5 and 24±1° C. Product formation was calculatedfrom the radioactive charcoal-binding assay.

Microtubule Destabilizing Drug Treatments

HEK 293T, HeLa, and HepG2 cells were treated for 6 hours with 25 μMcolchicines (Sigma) and lysed in low-stringency lysis buffer. Lysateswere equilibrated by total protein concentration and were separated on10% SDS-PAGE gels and western blotted as described above.

Results

Human SIRT2 Exhibits NAD-Dependent Histone Deacetylase Activity

To determine whether SIRT2 contained the NAD-dependent deacetylaseactivity associated with other SIR2 related proteins, E. coli purifiedrecombinant SIRT2 was incubated with increasing concentrations of NADand a peptide corresponding to the amino terminal tail of histone H4(a.a. 1-23) acetylated in vitro.

FIGS. 1A-D depict NAD-dependent deacetylation of a histone peptide byhuman SIRT2. FIG. 1A. The enzymatic activity of recombinant 6-His-SIRT2on a [³H]acetylated histone H4 peptide (a.a. 1-23) was measured in thepresence of increasing concentrations of NAD. Released acetate wasextracted and measured by scintillation counting. FIG. 1B. The enzymaticactivity of immunoprecipitated protein from FLAG or SIRT2-FLAGtransfected HEK 293T cells on a [³H] acetylated histone H4 peptide (a.a.1-23) was measured in the presence of increasing concentrations of NAD.10% of immunoprecipitated input into the enzymatic reaction was analyzedby SDS-PAGE and western blotting analysis with an anti-FLAG antibody.FIG. 1C. Similar reaction as described in (B) with SIRT2-FLAG −/+ NAD orHDAC6-FLAG and increasing concentrations of TSA. SDS-PAGE and westernblotting analysis as described in (B). FIG. 1D. Similar reaction asdescribed in (B) with SIRT2-FLAG −/+ NAD and increasing concentrationsof nicotinamide. SDS-PAGE and western blotting analysis as described in(B).

A dose dependent increase in histone deacetylase (HDAC) activity wasobserved in response to increasing concentrations of NAD (FIG. 1A). Afurther increase in NAD concentration to 10 mM resulted in a reductionin deacetylase activity (FIG. 1A). Similarly, a FLAG epitope-taggedSIRT2 protein (SIRT2-FLAG) was immunoprecipitated after transfection ofHEK 293T cells. SIRT2-FLAG showed a similar increase in HDAC activity inresponse to increasing concentrations of NAD from 1 μM to 1 mM (FIG.1B). However, this HDAC activity further increased in response to NADconcentrations of 10 mM (FIG. 1B). Equivalent amount of SIRT2-FLAG waspresent in each reaction as demonstrated by western blotting analysis(FIG. 1B).

Our results confirm that SIRT2 contains NAD-dependent histonedeacetylase activity as described previously (Tanner et al., 2000;Finnin et al., 2001). Furthermore, the observation that recombinantSIRT2 is enzymatically active indicates that the deacetylase activity ofSIRT2 does not require associated factors (FIG. 1A). However, thepresence of associated factors in cellular lysates could play a role inthe increased activity observed in the presence of high concentrationsof NAD (FIG. 1B).

In contrast to class I and class II deacetylases, class III deacetylasesare reported to be insensitive to the potent class I and class IIinhibitor Trichostatin A (TSA) (Yoshida et al., 1990; Furumai et al.,2001). To confirm that the SIRT2 enzymatic activity is insensitive toTSA, the enzymatic activity of immunoprecipitated SIRT2-FLAG with 1 mMNAD in the presence or absence of increasing concentrations of TSA(100-1600 nM) was measured. No change in HDAC activity of SIRT2 wasmeasured in response to increasing concentrations of TSA (FIG. 1C). Incontrast, HDAC6-FLAG immunoprecipitated from transfected HEK 293T waspotently inhibited by 400 nM TSA (FIG. 1C).

Nicotinamide represents the first product from hydrolysis of thepyridinium-N-glycosidic bond of NAD (Landry et al., 2000) and functionsas an effective inhibitor for the related SIRT1 protein (Luo et al.,2001). To test whether SIRT2 is also inhibited by nicotinamide,SIRT2-FLAG immunoprecipitated from transfected lysates was incubatedwith increasing concentrations of nicotinamide. A dose dependentdecrease in HDAC activity was observed with increasing concentrations ofnicotinamide ranging from 156 μM to 20 mM (FIG. 1D).

FIGS. 2A-C depict inactivation of SIRT2 histone deacetylase activity bypoint mutations within the SIRT2 catalytic domain. FIG. 2A. Schematicdiagram of Sir2 proteins from S. cerevisiae, C. elegans, and Drosophilaaligned with human SIRT2 within a region of the Sir2 domain (schematicadapted from (Frye, 1999)). Highlighted are two key residues necessaryfor histone deacetylase activity (Finnin et al., 2001). Genbankaccession numbers of proteins described are: S. cerevisiae NP 010242, C.elegans NP 501912, D. melanogaster AAC79684, and human SIRT2 NP 036369.FIG. 2B. The enzymatic activity of immunoprecipitated protein from FLAGvector and SIRT2-FLAG wild-type, N152A, or H187Y mutants transfected HEK293T cells on a [³H] acetylated histone H4 peptide (a.a. 1-23) wasmeasured with or without NAD. 10% of immunoprecipitation input into theenzymatic reaction was analyzed by SDS-PAGE and western blotting with ananti-FLAG antibody. FIG. 2C. The enzymatic activity ofimmunoprecipitated protein from GFP or GFP-SIRT2 wild-type, N152A, orH187Y mutants in assay as described in (B). SDS-PAGE and western blot asdescribed in (B) using an anti-GFP antibody.

The Sir2 proteins contain a highly conserved domain, the Sir2 domain,associated with enzymatic activity. A number of highly conservedresidues have been identified within this domain that are necessary fordeacetylase activity (highlighted in FIG. 2A) (Finnin et al., 2001). Toconfirm the role of these residues in deacetylase activity, asparagine168 was substituted with an alanine and histidine 187 with a tyrosineboth within the context of SIRT2-FLAG and in a fusion protein betweenGFP and the N terminus of SIRT2 (GFP-SIRT2). It was demonstrated thatboth proteins immunoprecipitated with either anti-FLAG of anti-GFP,respectively, contain NAD-dependent activity (FIG. 2B,C). In bothconstructs, the N168A and H187Y substitutions abolished this observedHDAC activity (FIG. 2B,C).

Sub-Cellular Distribution of Human SIRT2

While class I HDACs remain exclusively nuclear, class II HDACs shuttlebetween the nucleus and cytoplasm. This nucleocytoplasmic shuttling isregulated by phosphorylation and represents an element in the regulationof their enzymatic activity (Fischle et al., 2001). Interestingly, classIII HDACs exhibit variable sub-cellular distribution. Mouse Sir20α andits human homologue SIRT1 are localized primarily to the nucleus (Vaziriet al., 2001; Luo et al., 2001). In contrast, mouse and human SIRT2 arelocalized primarily to the cytoplasm (Afshar and Murnane, 1999; Yang etal., 2000), and interestingly the human SIRT3 protein is localized tomitochondria (Schwer et. al. submitted). The sub-cellular localizationof SIRT2, both as a fusion protein with GFP and as a FLAG-taggedprotein, was determined by two independent approaches. First, nuclearand cytoplasmic extracts from transfected HEK 293T cells were purified,and tested these extracts by western blotting with anti-GFP or anti-FLAGantibodies. GFP-SIRT2 was exclusively cytoplasmic whereas SIRT2-FLAG waspredominately cytoplasmic with a small fraction present within thenuclear compartment. Probing of the same fractions for a knowncytoplasmic protein, p65, and for a known nuclear protein, Lamin A,confirmed the purity of our cytoplasmic and nuclear fractions.

Second, immunofluoresence microscopy was used to further confirm thesub-cellular localization of SIRT2. After transfection of GFP-SIRT2 andSIRT2-FLAG into HeLa cells, an exclusively cytoplasmic localization forboth fusion proteins was observed. Furthermore, it was noted that SIRT2was locally more concentrated at an apolar axis of the nucleus. Thislocalization pattern coincided with the characteristic increase intubulin localization at the microtubule organization center (MTOC), alsolocated at an apolar axis of the nucleus.

Tubulin Deacetylation by Human SIRT2 In Vivo

The localization of SIRT2 in a region corresponding to the MTOC enticedus to test whether SIRT2 can deacetylate tubulin. Following transfectionof HeLa cells with GFP-SIRT2, which remains catalytically active as adeacetylase (FIG. 2C), cells were stained with an antiserum specific forα-tubulin acetylated at lysine-40 (Pipemo et al., 1987). It was foundthat cells expressing GFP-SIRT2 showed a marked reduction in acetylatedtubulin in comparison to neighboring untransfected cells. As a control,cells transfected with an expression vector for GFP alone exhibited noalteration in the level of acetylated tubulin. Two possibilities wereconsidered to explain these results. First, SIRT2 could be a bona fidetubulin deacetylase. Second, the active SIRT2 may regulate tubulinpolymerization dynamics thus resulting in reduced polymerizedmicrotubules, which may affect the acetylation state of α-tubulin. Toanswer this question, GFP-SIRT2 transfected cells were stained with anantisera that recognizes α-tubulin irrespective of the acetylationstate. No visible change in the microtubule network was observed incells transfected with GFP-SIRT2 in comparison to untransfected cells.These results are consistent with the model that SIRT2 is a functionaltubulin deacetylase.

To verify that the deacetylase activity of SIRT2 was necessary forα-tubulin deacetylation, GFP-SIRT2 expression vectors containing ourcatalytically inactive mutations N168A and H187Y were transfected intoHeLa cells. It was observed that catalytically inactive mutant SIRT2 hadno effect on the level of acetylated α-tubulin. These results indicatethat expression of wild-type SIRT2 in vivo leads to the deacetylation oflysine-40 on α-tubulin, mediated by the deacetylase activity of SIRT2.

Human SIRT2 Deacetylates Tubulin In Vitro

To test directly the ability of SIRT2 to deacetylate α-tubulin, an exvivo tubulin deacetylation assay was developed. In this assay, HEK 293Tcells were transfected with SIRT2-FLAG followed by immunoprecipitationof the FLAG-tagged protein (FIG. 3A). The immunoprecipitated materialwas separated into two fractions. The first fraction was used to measureHDAC activity using the acetylated histone H4 peptide. The secondfraction was used for a tubulin deacetylation activity assay (FIG. 3A)using total cellular lysates from untransfected HEK 293T cells assubstrate for acetylated α-tubulin. The extent ofacetylation/deacetylation of α-tubulin was determined by westernblotting analysis using a specific antisera for acetylated α-tubulin.First, it was demonstrated that material immunoprecipitated aftertransfection of HEK 293T cells with an empty FLAG vector has no effecton tubulin acetylation (FIG. 3B, lanes 1). This result demonstrates thatneither the lysate utilized as acetylated α-tubulin substrate nor theimmunoprecipitation procedure carry any significant levels of tubulindeacetylase activity. In contrast, incubation of cellular lysate withthe immunoprecipitated SIRT2-FLAG protein deacetylated tubulin in anNAD-dependent manner (FIG. 3B, lanes 2 and 3). In addition, it wasconfirmed that the catalytically inactive mutants N168A and H187Y do notdeacetylate α-tubulin (FIG. 3B, lanes 4 and 5).

Humans contain seven highly conserved proteins with homology to S.cerevisiae Sir2p (Frye, 1999; Frye, 2000). To determine whetherα-tubulin deacetylation activity was restricted to SIRT2, all sevenhuman Sir2 proteins tagged with FLAG at the C-terminus were expressed inHEK 293T cells and immunoprecipitated. The immunoprecipitated materialwas tested both in our ex vivo tubulin deacetylation assay and in HDACactivity assay using the histone H4 peptide. Of the seven SIRT proteins,only SIRT1, 2 and 3 demonstrated significant HDAC activity on a histoneH4 peptide (FIG. 3C). SIRT4,5,6, and 7 had no detectable HDAC activity(FIG. 3C). In contrast, only SIRT2 deacetylated tubulin in vitro (FIG.3C). These results demonstrate that SIRT2 is the only class IIIdeacetylase protein capable of deacetylation of tubulin.

Treatment of cells with the class I and class II HDAC inhibitor TSAinduces hyperacetylation of α-tubulin (Grozinger et al., 2001). Thisresult suggest that a class I or class II HDAC can also deacetylateα-tubulin. Since class I HDACs are exclusively nuclear, the class IIHDACs, which are know to shuttle between the nucleus and cytoplasm, werefocused on. HDAC4,5,6, and 7 FLAG-tagged at the C-terminus, weretransfected in HEK 293T, immunoprecipitated and tested both for HDACactivity and for tubulin deacetylase activity. All class II HDACs showedabundant deacetylase activity on the acetylated H4 peptide (FIG. 3D).However, none of the class II HDACs tested contained tubulin deacetylaseactivity (FIG. 3D).

Purification of a SET3 complex from S. cerevisiae has identified bothclass I and class III HDACs present in the same multi-protein complex(Pijnappel et al., 2001). To confirm that a SIR2-like protein is thesole component in the immunoprecipitation responsible for tubulindeacetylation, it was tested whether nicotinamide could inhibit thetubulin deacetylase activity associated with SIRT2. HEK 293T cells weretransfected with SIRT2-FLAG or the empty vector as a control, followedby immunoprecipitation using anti-FLAG. No deacetylation was noted inthe absence of NAD (FIG. 3E, lanes 2). Addition of NAD to the SIRT2-FLAGreaction led to the complete deacetylation of tubulin. This reaction wascompletely inhibited in the presence of 5 mM nicotinamide (FIG. 3E,lanes 3 and 4). As a control, Trichostatin A, a potent inhibitor ofclass I and class II HDACs, had no effect on tubulin deacetylation bySIRT2 (FIG. 3E, lane 5). These results confirm that the sole tubulindeacetylase found within the immunoprecipitated SIRT2 material is SIRT2.

FIGS. 3A-E depict SIRT2 tubulin deacetylates tubulin ex vivo. FIG. 3A.Schematic diagram of ex vivo tubulin deacetylation assay. FIG. 3B.Immunoprecipitated protein corresponding to SIRT2-FLAG wild-type, N168A,or H187Y, was incubated with cellular lysate in vitro. The reactionproducts were separated by SDS-PAGE and western blotting using specificantisera for acetylated tubulin, tubulin and FLAG. One half ofimmunoprecipitation was subjected to HDAC activity assay using a histoneH4 peptide acetylated in vitro. FIG. 3C. Similar tubulin deacetylationassay using the seven class III HDACs, SIRT1-7-FLAG, as described for(B). One half of immunoprecipitation was subjected to HDAC activityassay as described for (B). FIG. 3D. Similar tubulin deacetylation assayusing the class II HDACs as described for (B). One half ofimmunoprecipitation was subjected to HDAC activity assay as describedfor (B). FIG. 3E. Inhibition of SIRT2 tubulin deacetylation was examinedusing the similar tubulin deacetylation assay with SIRT2-FLAG as in (B)including reactions incubated with either 5 mM nicotinamide or 400 nMTSA.

Enzymatic Kinetics of Human SIRT2 and Yeast Hst2p

To further analyze the deacetylation of tubulin by SIRT2, a detailedenzymatic analysis was performed. For comparison, the highly activeyeast histone deacetylase Hst2p was analyzed alongside the human enzyme.Hst2p exhibits strong selectivity for peptides corresponding to histoneH3 acetylated on lysine-14 (Tanner et al., 2000; Landry et al., 2000). A9-amino acid synthetic α-tubulin peptide (MPSD(AcK)TIGG; SEQ ID NO:08),acetylated on lysine-40 and a 20-amino acid synthetic histone H3 peptide(ARTKQTARKSTGG(AcK)APRKQL; SEQ ID NO:03), acetylated on lysine-14, wereutilized to measure initial velocities of Hst2p and SIRT2 at varioushistone H3 or tubulin peptide concentrations. The resulting saturationcurves were fitted to the Michaelis-Menten equation, yielding thekinetic parameters k_(cat), K_(m), and V/K. The K_(cat) value is themaximal rate of enzyme turnover when substrates are at saturatingconcentrations. The K_(m) value is the concentration of substrate neededto reach ½ the maximal velocity. The most physiologically relevantconstant is the V/K value, as this second-order constant defines therate of the reaction when substrate concentrations are not at saturatinglevels, and reflects both substrate binding and catalysis. Becausecellular enzymatic reactions rarely occur under maximal velocityconditions (i.e. saturating substrate levels), dramatic differences inthe V/K value of the enzyme will likely reflect the most relevant invivo consequences.

With acetylated tubulin peptides as substrate, SIRT2 exhibited astriking preference for this substrate relative to yeast Hst2p (FIG.4A). This difference was −60-fold and was reflected in both the k_(cat)and V/K values, which were 0.144±0.005 s⁻¹ and 894±100 M⁻¹s⁻¹ for SIRT2,and 0.00254±0.0003 s⁻¹ and 14.9 M⁻¹ s⁻¹ for Hst2p, respectively. Incontrast, when acetylated H3 peptide was employed as substrate, Hst2pdemonstrated a ˜200-fold stronger preference for H3 peptide relative toSIRT2 (FIG. 4B). These differences were reflected in the V/K and K_(m)values, which were 3930±261 M⁻¹ s⁻¹ and 54.2±3.6 μM for SIRT2, and717,900±35,900 M⁻¹s⁻¹ and 0.280±0.014 μM for Hst2p, respectively. Thek_(m) values (˜0.2 s⁻¹) were similar between the two enzymes.

These results indicate that the Sir2-family of NAD-dependentdeacetylases display remarkable differences in substrate specificity,and that human SIRT2 displays a marked preference for the acetylatedtubulin peptide relative to the yeast enzyme Hst2p (FIGS. 4A, C, and D).To provide further evidence that SIRT2 has reduced capacity todeacetylate histones in comparison to Hst2p, the ability of SIRT2 andHst2p to deacetylate core histones was examined. Purified histonesacetylated in vitro by PCAF were incubated with catalytic amounts ofeither SIRT2 or Hst2p, and deacetylation was quantified (FIG. 4C). Theapparent V/K values were determined and compared. Consistent with thepeptide results, Hst2p exhibited a 7-fold higher V/K value than SIRT2.

FIGS. 4A-D depict the substrate preference for SIRT2. FIG. 4A. Initialvelocities measured at varying concentrations of tubulin peptide,(MPSD(AcK)TIGG) for SIRT2 (open circles) and for Hst2p (closed circles)with concentrations and conditions described in materials and methods.The curve with SIRT2 represents the average rates from 3 differentexperiments. The Hst2p curve is a representative data set from 1 of 3separate experiments. The indicated NAD concentrations are saturatingwith respect to each enzyme.

FIG. 4B. Initial velocities for each enzyme measured at varyingconcentrations of acetylated H3 peptide, (ARTKQTARKSTGG(AcK)APRKQL; SEQID NO:03) for SIRT2 (open circles) and for Hst2p (closed circles) withconcentrations and conditions described in materials and methods. Theindicated NAD concentrations are saturating with respect to each enzyme.

FIG. 4C. Kinetic progress curves of histone deacetylation by SIRT2 andHst2p. Either SIRT2 (50 nM) or Hst2p (20 nM) were incubated with ˜1.2 μMPCAF-acetylated calf thymus core histones.

FIG. 4D. Table listing the results when the graphs from (A) and (B) werefitted to the Michaelis-Menten equation to obtain the kinetic parametersof K_(m), k_(cat), and V/K.

Regulation of MIZ-1 Microtubule Binding by α-Tubulin Acetylation

A recent report documented the sequestration of the transcriptionfactor, Myc-interacting zinc finger 1 (MIZ-1), by binding to tubulin onpolymerized microtubules (Ziegelbauer et al., 2001). In this report, theauthors suggest that upon depolymerization of the microtubule network,MIZ-1 is released by its binding tubulin and is free to translocate tothe nucleus. Interestingly when the polymerization state of tubulin wasinterrogated by treatment with either colchicine or nocodazole, tubulindeacetylation in response to depolymerization by both treatments wasobserved (FIG. 5A). In addition, It was observed that only polymerizedmicrotubules can serve as a substrate for acetylation of α-tubulin bythe yet undefined tubulin acetyltransferase. In their report,Ziegelbauer et al., demonstrate that in the hepatocellular carcinomacell line, HepG2, MIZ-1 has a predominately cytoplasmic localization.Interestingly, when the localization of GFP-MIZ-1 in HeLa cells, whichhave a low level of acetylated α-tubulin, was examined, it was foundthat it is predominately localized to the nucleus. However, when thesecells were treated with TSA, which leads to hyperacetylation of tubulin,a distinct shift in localization of GFP-MIZ-1 from the nucleus to thecytoplasm was observed.

To determine if this relocalization of MIZ-1 to the cytoplasm upontreating cells with TSA is dependent on the acetylation state ofα-tubulin, the deacetylase activity of SIRT2 was utilized. Uponco-transfection of cells with GFP-MIZ-1 and either the FLAG vector,SIRT2-FLAG wild-type or the catalytically inactive N168A mutant ofSIRT2-FLAG, it was noticed that in all cases GFP-MIZ-1 localizedpredominately to the nucleus (FIG. 5B). However upon treatment of thesecells upon TSA, a statistically significant relocalization of GFP-MIZ-1into the cytoplasm was seen when GFP-MIZ-1 was co-transfected witheither the FLAG empty vector or the catalytically inactive SIRT2-FLAGN168A of 36.9% and 35.3%, respectively (FIG. 5B). However when theGFP-MIZ-1 was co-transfected with SIRT2-FLAG wild-type a significantdecrease in percentage of cells with GFP-MIZ-1 localized in thecytoplasm to 11.2% was seen (FIG. 5B). In their report, Ziegelbauer etal., demonstrate co-localization of MIZ-1 with the microtubule networkin HepG2 cells, where MIZ-1 is apparently localized predominately in thecytoplasm.

As demonstrated above, in HeLa cells, GFP-MIZ-1 is localized primarilyin the nucleus. To rule out the possibility is that the GFP-taggedversion of MIZ-1 was altering MIZ-1 ability to bind to tubulin thesub-cytoplasmic localization in cells co-transfected with GFP-MIZ-1 andempty FLAG vectors and treated with TSA was assayeds. A colocalizationof GFP-MIZ-1 with acetylated tubulin was visualized by confocalmicroscopy. This colocalization indicates that GFP-MIZ-1 maintains theability to bind to tubulin, and indicates that upon treatment oftransfected cells with TSA, GFP-MIZ-1 will translocate from the nucleusto the cytoplasm where it will bind to the hyperacetylated microtubulenetwork. These data suggest that MIZ-1 sub-cellular distribution isregulated not only by the state of tubulin polymerization, but also bythe acetylation state of α-tubulin within the microtubule network.

FIGS. 5A and 5B depict regulation of MIZ-1 sub-cellular distribution byacetylated tubulin.

FIG. 5A. HeLa, 293T and HepG2 cells were treated with 10 μg/mLcolchicine or 1 μg/mL nocodazole for 6 hours. Cells were harvested andlysates separated by SDS-PAGE and western blotting using specificantisera for acetylated tubulin and tubulin.

FIG. 5B. Transfected cells were counted as either GFP-MIZ-1 localizedcompletely nuclear or with partial cytoplasmic retention. Cell countswere derived from inspection of at least 150 transfected cells from sixmicroscopic fields. Results are average of three separate transfectionswith error bars representing standard deviation between eachtransfection. Data set is representative of three independentexperiments.

Example 2 Inhibition of SIRT2

Based on the dependency of SIRT proteins on NAD, a number of moleculeswith structure homology to NAD were tested as potential inhibitors oftheir enzymatic activity. The previously characterized molecule of1-β-D-Ribofuranosyl-1-2-4-triazole-3-carboxamide (Ribavirin) has beenused as an approved antiviral agent against hepatitis C and is an analogof the nicotinamide portion of NAD.

Inhibitory activity of ribavirin against SIRT2 and SIRT3 proteins wastested using recombinant SIRT2 proteins and an in vitro assay. Ribavirinwas incubated with recombinant protein only in buffer for 10 minutes;then NAD and substrate were added to start the reaction. Reactions wereincubated for 2 hours at room temperature. Reactions were stopped byadding 25 μL stop buffer (0.1M HC1, 0.16M acetic acid) and vortexingbriefly. Ethyl acetate extraction isolated any liberated acetyl groups.Ethyl acetate (0.5 ml) was added, mixture was vortexed for 15 secondsand spun at 14,000 rpm for 5 minutes. The upper phase (0.4 ml) was addedto 5 mL scintillation fluid (Econofluor-2; Packard) and counted.

The results are shown in FIG. 7. Ribavirin inhibited SIRT2 in vitro HDACactivity with an approximate 50% inhibition at 25 μM, but had no effecton SIRT3 in vitro HDAC activity. This experiment demonstrates theidentification of a novel and specific inhibitor for SIRT2.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of inhibiting human sirtuin type 2 (SIRT2) tubulindeacetylase activity in a subject, the method comprising administeringto the subject an agent that inhibits human SIRT2 sirtuin type 2 (SIRT2)tubulin deacetylase enzymatic activity, wherein the agent isadministered in an amount effective to reduce SIRT2 tubulin deacetylaseenzymatic activity by at least 10% relative to the level of SIRT2tubulin deacetylase enzymatic activity in the absence of the agent,wherein the agent comprises ribavirin.
 2. The method of claim 1, whereinthe ribavirin is administered to the subject by oral administration. 3.The method of claim 1, wherein the ribavirin is administered to thesubject at a dose of from about 30 mg to about 1200 mg per day.
 4. Themethod of claim 1, wherein the subject has cancer.
 5. The method ofclaim 4, wherein the cancer is a carcinoma, a sarcoma, a leukemia, or alymphoma.
 6. The method of claim 4, wherein the agent that inhibitsSIRT2 tubulin deacetylase enzymatic activity reduces the level or rateof cell proliferation by at least 10% relative to the level or rate ofcell proliferation in the absence of the agent.
 7. The method of claim4, wherein the agent that inhibits SIRT2 tubulin deacetylase enzymaticactivity reduces tumor mass by at least 10% relative to the tumor massin the absence of the agent.
 8. The method of claim 1, wherein the agentthat inhibits SIRT2 tubulin deacetylase enzymatic activity isadministered in combination with a second therapy.
 9. The method ofclaim 8, wherein the second therapy comprises chemotherapeutictreatment.
 10. The method of claim 9, wherein the chemotherapeutictreatment comprises a cytotoxic or a cytostatic agent.
 11. The method ofclaim 9, wherein the chemotherapeutic treatment comprises achemotherapeutic agent selected from: an alkylating agent; anitrosourea; an antimetabolite; an antitumor antibiotic; a vincaalkaloid; and a steroid hormone.
 12. The method of claim 8, wherein theagent that inhibits SIRT2 tubulin deacetylase enzymatic activity isadministered as an adjuvant to a standard cancer therapy.
 13. The methodof claim 8, wherein the second therapy comprises treatment with anantineoplastic agent.
 14. The method of claim 13, wherein theanti-neoplastic agent comprises an antineoplastic agent selected from:an alkylating agent; an antimetabolite; a vinca alkyloid; anantineoplastic antibiotic; a platinum complex; a tyrosine kinaseinhibitor; an inhibitor of a receptor tyrosine kinase that is involvedin a growth factor signaling pathway; an inhibitor of a non-receptortyrosine kinase involved in a growth factor signaling pathway; aserine/threonine kinase inhibitor; an inhibitor of a kinase involved incell cycle regulation; a taxane; a tumor-associated antigen antagonist;and a tumor growth factor antagonist.